Effects of 3-dimensional culture conditions (collagen-chitosan nano-scaffolds) on maturation of dendritic cells and their capacity to interact with T-lymphocytes

In the body, there is a natural three-dimensional (3D) microenvironment in which immune cells, including dendritic cells (DC), play their functions. This study evaluated the impact of using collagen-chitosan 3D nano-scaffolds in comparisons to routine 2D culture plates on DC phenotype and functions. Bone marrow-derived DC were cultured on scaffolds and plates and then stimulated with lipopolysaccharide (LPS) or chitosan-based nanoparticles (NP) for 24?h. Thereafter, DC viability, expression of maturation markers and levels of cytokines secretion were evaluated. In another set of studies, the DC were co-cultured with allogenic T-lymphocytes in both the 2D and 3D systems and effects on DC-induction of T-lymphocyte proliferation and cytokine release were analyzed. The results indicated that CD40, CD86 and MHC II marker expression and interleukin (IL)-12, IL-6 and tumor necrosis factor (TNF)-α secretion by DC were enhanced in 3D cultures in comparison to by cells maintained in the 2D states. The data also showed that DNA/chitosan NP activated DC more than LPS in the 3D system. T-Lymphocyte proliferation was induced to a greater extent by DNA/NP-treated DC when both cell types were maintained on the scaffolds. Interestingly, while DC induction of T-lymphocyte interferon (IFN)-γ and IL-4 release was enhanced in the 3D system (relative to controls), there was a suppression of transforming growth factor (TGF)-β production; effects on IL-10 secretion were variable. The results here suggested that collagen-chitosan scaffolds could provide a pro-inflammatory and activator environment to perform studies to analyze effects of exogenous agents on the induction of DC maturation, NP uptake and/or cytokines release, as well as for the ability of these cells to potentially interact with other immune system cells in vitro.     

Fabrication and Characterization of Waterborne Biodegradable Polyurethanes 3-Dimensional Porous Scaffolds for Vascular Tissue Engineering

In this study, a series of 3-D interconnected porous scaffolds with various pore diameters and porosities was fabricated by freeze-drying with non-toxic biodegradable waterborne polyurethane (WBPU) emulsions of different concentration. The structures of these porous scaffolds were characterized by scanning electron microscopy (SEM), and the pore diameters were calculated using CIAS 3.0 software. The pores obtained were 3-D interconnected in the scaffolds. The scaffolds obtained at different pre-freeze temperatures showed a pore diameter ranging from 2.8 to 99.9 μm with a pre-freezing temperature of ?60°C and from 13.1 to 229.1 μm with a pre-freezing temperature of ?25°C. The scaffolds fabricated with WBPU emulsions of different concentration at the same pre-freezing temperature (?25°C) had pores with mean pore diameter between 90.8 and 39.6 μm and porosity between 92.0 and 80.0%, depending on the emulsion concentration. The effect of porous structure of the scaffolds on adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) cultured in vitro was evaluated using the MTT assay and environmental scanning electron microscopy (ESEM). It was found that the better adhesion and proliferation of HUVECs on 3-D scaffolds of WBPU with relative smaller pore diameter and lower porosity than those on scaffolds with larger pore and higher porosity and film. Our work suggests that fabricating a scaffold with controllable pore diameter and porosity could be a good method to be used in tissue-engineering applications to obtain carriers for cell culture in vitro.     

Tissue engineering scaffolds containing embedded fluorinated-zeolite oxygen vectors

Efficient oxygen supply is a continuing challenge for the fabrication of successful tissue engineered constructs with clinical relevance. In an effort to enhance oxygen delivery we report the feasibility of using fluorinated zeolite particles embedded in three-dimensional (3-D) polyurethane scaffolds as novel oxygen vectors. First, 1H,1H,2H,2H-perfluorodecyltriethoxysilane was successfully coupled to zeolite framework particles to examine the dose-dependent dissolved oxygen concentration. Following this, the fluorinated-zeolite (FZ) particles were embedded in 3-D tissue engineering polyurethane scaffolds. Our data demonstrates an even distribution of FZ particles in the 3-D scaffolds without affecting the scaffold porosity or pore size. Human coronary artery smooth muscle cell (HCASMC) proliferation on FZ-containing polyurethane (PCU-FZ) scaffolds was significantly greater than on control scaffolds (P = 0.05). Remarkably, cell infiltration depths on the PCU-FZ scaffolds was double that on PCU control scaffolds. Taken together, our data suggest the potential of PCU-FZ scaffolds for tissue engineering with enhanced oxygen delivery to cells.     

Thermal dehydration treatment and glutaraldehyde cross-linking to increase the biostability of collagen–chitosan porous scaffolds used as dermal equivalent

A biodegradable scaffold for skin-tissue engineering was designed using collagen and chitosan, which are common materials for biomedical application. The scaffolds containing different amounts of chitosan were prepared by mixing the collagen and chitosan solutions followed by removal of the solvent using a freeze-drying method. The cross-linking treatment of these scaffolds was performed using the dehydrothermal treatment (DHT) method or glutaraldehyde (GA) to increase their biostability. The effect of the chitosan concentration and the cross-linking methods on the morphology of these scaffolds was studied by SEM. The water retention and the biodegradability in vitro of various collagen-chitosan scaffolds were investigated. Finally the biocompatibility of the collagen-chitosan (10 wt% chitosan) scaffold treated with different cross-linking methods was evaluated using a in vivo animal test. A mild inflammatory reaction could be detected in the early stages, and GA treatment can decrease the inflammatory reaction in a long-term implantation. After implantation for four weeks, all kinds of scaffolds, especially the GA-treated scaffolds (Col-GA) were filled with a large number of fibroblasts and were vascularized to a certain extent. These results suggest that the GA-treated scaffold has an increased biostability and excellent biocompatibility. It can be a potential candidate for skin-tissue engineering.     

Cryopreservable and tumorigenic three-dimensional tumor culture in porous poly(lactic-co-glycolic acid) microsphere

In vitro tumor models that mimic in vivo conditions may be ideal for screening anticancer drugs and their formulations and developing tumors in animal models. Three-dimensional (3-D) culture of cancer cells on polymeric scaffolds can be an option for such models. In the present study, porous poly(lactic acid-co-glycolic acid) (PLGA) microsphere was used both as a cancer cell culture substrate to expand cells and as a cancer cell transplantation vehicle for tumor construction in mice. MCF-7 cells cultured on porous PLGA microspheres in stirred suspension bioreactors expanded by 2.8-fold over seven days and maintained viability. At three months after inoculation with 2 × 10⁶ cells/site, the tumor formation by MCF-7 cells cultured on microspheres was much more effective (4 tumors/5 mice) than its counterpart cultured on plates (1/5). More importantly, cell viability and metabolic activity were not significantly changed even after one freeze–thaw cycle of the 3-D culture. MCF-7 cells cultured on the microspheres and the cells in 3-D after cryopreservation were more resistant to doxorubicin than MCF-7 cells cultured on plates.     

The effect of vitronectin on the differentiation of embryonic stem cells in a 3D culture system

While stem cell niches in vivo are complex three-dimensional (3D) microenvironments, the relationship between the dimensionality of the niche to its function is unknown. We have created a 3D microenvironment through electrospinning to study the impact of geometry and different extracellular proteins on the development of cardiac progenitor cells (Flk-1⁺) from resident stem cells and their differentiation into functional cardiovascular cells. We have investigated the effect of collagen IV, fibronectin, laminin and vitronectin on the adhesion and proliferation of murine ES cells as well as the effects of these proteins on the number of Flk-1⁺ cells cultured in 2D conditions compared to 3D system in a feeder free condition. We found that the number of Flk-1⁺ cells was significantly higher in 3D scaffolds coated with laminin or vitronectin compared to colIV-coated scaffolds. Our results show the importance of defined culture systems in vitro for studying the guided differentiation of pluripotent embryonic stem cells in the field of cardiovascular tissue engineering and regenerative medicine.     

Immunomodulatory effects of the methanolic extract from Pouteria campechiana leaves in macrophage functions

The present study aimed to examine the immunomodulatory properties of the methanolic (MeOH) extract from Pouteria. campechiana leaves in peritoneal macrophages of Balb/c mice. Peritoneal macrophages isolated from mice and Vero cells were treated with the MeOH extract from leaves. Cell viability of the macrophages and Vero cells were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide method. The phagocytic activity, as nitric oxide (NO), hydrogen peroxide (H₂O₂), interleukin 6 (IL-6) and tumour necrosis factor α (TNF-α) production were evaluated on peritoneal macrophages. Results showed that the MeOH extract from leaves was able to stimulate the phagocytic activity and increase NO, H₂O₂ and cytokines production. The viability assays do not show cytotoxic effect on cell viability and cause a significative proliferative effect in the macrophages of a concentration-dependent manner. These results conclude that the MeOH extract from P. campechiana leaves possessed a stronger immunostimulatory effect in a concentration-dependent manner without affect the cell viability.     

Immunotropic activity of plant extract echinosol

The formulation of medicinal herbal tea Echinosol was developed. Immunotropic properties of aqueous-alcohol extract Echinosol were studied. This extract (300 mg/kg) had a stimulatory effect on the following parameters: formation of T cell precursors and production of T cell effectors; migration of lymphocytes, monocytes and neutrophils into the blood; digestive activity of neutrophils; migration of monocytes and lymphocytes into the peritoneal exudate; expression of C_3b receptors on macrophages; phagocytic capacity of cells due to the existence of C_3b receptors; number of antibody-producing cells in the spleen; and synthesis of immunoglobulins. Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 8, pp. 188–191, August, 2008     

Effect of macrophage colony-stimulating factor (M-CSF) on macrophage morphology,phagocytosis, and intracellular multiplication of Histoplasma capsulatum

M-CSF induced dramatic morphological changes in resident peritoneal macrophages (R-PM) in a time and dose dependent manner. Macrophages increased 2–3-fold in size and developed a dendritic morphology. Compared to macrophages cultured in medium alone M-CSF-treated macrophages were less phagocytic (67 versus 82%) for serum opsonized yeast cells of Histoplasma capsulatum. However, in M-CSF macrophages the intracellular multiplication of ingested yeast cells were significantly inhibited compared to growth in control macrophages. The fungistatic activity of M-CSF macrophages persisted for at least 48 h after infection.     

Engineering vascularized soft tissue flaps in an animal model using human adipose–derived stem cells and VEGF+PLGA/PEG microspheres on a collagen-chitosan scaffold with a flow-through vascular pedicle

Insufficient neovascularization is associated with high levels of resorption and necrosis in autologous and engineered fat grafts. We tested the hypothesis that incorporating angiogenic growth factor into a scaffold–stem cell construct and implanting this construct around a vascular pedicle improves neovascularization and adipogenesis for engineering soft tissue flaps. Poly(lactic-co-glycolic-acid/polyethylene glycol (PLGA/PEG) microspheres containing vascular endothelial growth factor (VEGF) were impregnated into collagen-chitosan scaffolds seeded with human adipose-derived stem cells (hASCs). This setup was analyzed in vitro and then implanted into isolated chambers around a discrete vascular pedicle in nude rats. Engineered tissue samples within the chambers were harvested and analyzed for differences in vascularization and adipose tissue growth. In vitro testing showed that the collagen-chitosan scaffold provided a supportive environment for hASC integration and proliferation. PLGA/PEG microspheres with slow-release VEGF had no negative effect on cell survival in collagen-chitosan scaffolds. In vivo, the system resulted in a statistically significant increase in neovascularization that in turn led to a significant increase in adipose tissue persistence after 8 weeks versus control constructs. These data indicate that our model—hASCs integrated with a collagen-chitosan scaffold incorporated with VEGF-containing PLGA/PEG microspheres supported by a predominant vascular vessel inside a chamber—provides a promising, clinically translatable platform for engineering vascularized soft tissue flap. The engineered adipose tissue with a vascular pedicle could conceivably be transferred as a vascularized soft tissue pedicle flap or free flap to a recipient site for the repair of soft-tissue defects.     

Effects of trabecular calcium phosphate scaffolds on stress signaling in osteoblast precursor cells

The objective of this research was to investigate stress-signaling patterns in response to two-dimensional (2-D) and three-dimensional (3-D) calcium phosphate (CP) materials using human embryonic palatal mesenchyme cells (HEPM, CRL-1486, ATCC, Manassas, VA), an osteoblast precursor cell line. Control discs and scaffolds were fabricated from hydroxyapatite and beta tri-CP ceramics. Phospho-specific antibody cell-based ELISA technique was utilized on members of the mitogen-activated protein kinase cascade including; the extracellular signal-regulated kinases (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and the anti-apoptosis mediator protein kinase B (AKT). Quantification of these signals was evaluated during the early attachment phase of osteoblast precursor cells. In this study, it was observed that 3-D CP scaffolds significantly activated the stress mediators p38 and JNK but not ERK1/2. This signal trend was matched with an up-regulation in AKT, suggesting the ability of cells to manage high stress signals in response to 3-D CP architecture and that 3-D CP scaffolds are necessary for studies simulating a natural trabecular bone organization. The absence of these signals in 2-D CP surfaces indicated the importance of local architecture conditions on cell stress response. It was concluded from this study that osteoblast precursor cells cultured in 3-D CP scaffolds experience greater stress-signaling patterns when compared to 2-D CP surfaces.     

The pathologic cycle of the infection of the microsporidian Microgemma ovoidea (Thèl., 1895) Amigó et al. 1996 in the liver of the Red Band Fish (Cepola macrophthalma L.)

Pathologic study of the lesions caused by Microgemma ovoidea has shown that after the formation of the xenoma (stage 1), the parasitized cell is infiltrated by host macrophages (stage 2) and quickly encysted by the activity of fibroblasts that form a xenoma wall composed of collagenous fibers (stage 3). The phagocytic activity of the macrophages leads to the formation of a granuloma (stage 4) in which the cyst contents comprise macrophages filled with phagocytosed spores. This phagocytic activity is limited by the fact that some parts of the microsporidian spores, such as the spore walls, cannot be lysed by macrophages, which leads to the formation of fused giant cells containing nondigestible spore remnants. The final step in the process is healing (stage 5), in which some cells may start proliferating to regenerate the damaged area. Nevertheless, the host occasionally fails to control M. ovoidea infections. This failure can take two forms: bursting of the granuloma, or the appearance of secondary infections in granulomas, probably through parasitism of macrophages. Received: 2 April 1997 / Accepted: 4 June 1997     

In vitro assessment of three-dimensionally plotted nagelschmidtite bioceramic scaffolds with varied macropore morphologies

It is known that porous scaffolds play an important role in bone/periodontal tissue engineering. A new nagelschmidtite (NAGEL, Ca₇Si₂P₂O₁₆) ceramic has recently been prepared which shows excellent apatite mineralization ability and osteo-/cementostimulation properties in vitro. However, up to now porous NAGEL scaffolds have not been developed yet. There has been no systematic study of the effect of macropore morphology of bioceramic scaffolds on their physico-chemical and biological properties. The aim of this study was to prepare NAGEL scaffolds for bone tissue engineering applications. We applied a modified three-dimensional (3-D) plotting method to prepare highly controllable NAGEL scaffolds and investigated the effect of macropore morphology on the physico-chemical and biological properties. The results showed that the macropore size and morphology of 3-D plotted NAGEL scaffolds could be effectively controlled. Compared with β-tricalcium phosphate (β-TCP) scaffolds NAGEL scaffolds possess a significantly enhanced compressive strength, a higher modulus and better degradability. Nagel scaffolds with a square pore morphology presented a higher compressive strength, a higher modulus and greater weight loss rate than those with triangular and parallelogram pore morphologies. In addition, all of the NAGEL scaffolds with the three macropore morphologies supported the attachment and proliferation of MC3T3 cells. The proliferation of MC3T3 cells on NAGEL scaffolds with triangular and parallelogram structures was higher than that on β-TCP scaffolds with the same pore structure. Cells on all three groups of NAGEL scaffolds revealed higher alkaline phosphatase (ALP) activity compared with cells on β-TCP scaffolds, and among the three NAGEL scaffolds groups those with a parallelogram pore structure showed the highest ALP activity. Furthermore, the angiogenic cell experiments showed that the ionic products from NAGEL scaffolds promoted tube formation, expression of pro-angiogenic factors and their receptors on human umbilical vein endothelial (HUVECs) compared with β-TCP scaffolds, indicating that NAGEL scaffolds possessed improved angiogenesis capacity. Our results suggest that 3-D plotted NAGEL scaffolds are a promising bioactive material for bone tissue engineering by virtue of their highly controllable macropore structure, excellent mechanical strength, degradability and in vitro biological response to osteogenic/angiogenic cells.     

Over-expression of integrin β3 can partially overcome the defect of integrin β3 signaling in transglutaminase 2 null macrophages

Transglutaminase 2 (TG2) is a protein crosslinking enzyme with many additional biological functions. We have previously shown that in TG2^−/− mice the in vivo clearance of apoptotic cells is defective leading to autoimmunity. TG2 contributes to the formation of phagocytic portals by binding to both integrin β₃, a known phagocytic receptor, and its bridging molecule, MFG-E8. In TG2 null macrophages integrin β₃ cannot accumulate around the apoptotic cells and its signaling is impaired. In the present study we describe a subline of TG2 null mice, in which a compensatory increase in integrin β₃ expression, which resulted alone in a high receptor concentration around the apoptotic cells without the requirement for accumulation, partially corrected the defect in integrin β₃ signaling. Our data provide a proof for the concept that the function of TG2 is to stabilize accumulated integrin β₃ concentration in the phagocytic cup.     

Phenotypical analysis of adult rat olfactory ensheathing cells on 3-D collagen scaffolds

Olfactory ensheathing cell (OEC) transplantation is a promising or potential therapy for spinal cord injury (SCI). However, the effects of injecting OECs directly into SCI site have been limited and unsatisfied due to the complexity of SCI. To improve the outcome, proper biomaterials are thought to be helpful since these materials would allow the cells to grow three-dimensionally and guide cell migration. In this paper, we have studied the behavior of OECs in two-dimensional (2-D) condition as well as on three-dimensional (3-D) collagen scaffolds by analyzing their phenotypes such as cell proliferation, apoptosis, morphology, and gene activities of some neurotrophic factors and myelin proteins. OECs proliferation rate was increased on 3-D collagen scaffolds compared to the 2-D culture condition. OECs on 3-D collagen scaffolds also showed less apoptosis. In addition, OECs on 3-D collagen scaffolds maintained the original spindle-shape morphology and P75NTR gene activity. NGF, BDNF, and PLP were found to be upregulated in OECs cultured on 3-D collagen scaffolds by the semi-quantitative RT-PCR approach. The results suggested that 3-D collagen scaffolds provide suitable environments for the OECs to maintain their morphology as well as several important functional phenotypes and all these could be helpful for the effective treatment of SCI.     

Low-Fluence Photodynamic Treatment Modifies Functional Properties of Vascular Cell Wall

We studied the effect of low-fluence photodynamic treatment with Photosence preparation on functional properties of macrophages and endothelium in vitro. It was shown that low-intensity photodynamic treatment did not affect viability of these cells. Exposure (0.25 J/cm²) of endothelial cells loaded with Photosence did not change the expression of adhesion molecules, but reduced adhesion of peripheral blood mononuclears to these cells. The same low-intensity exposure inhibited phagocytic activity of macrophages and reduced activity of matrix metalloproteinase-9 produced by them.     

In vivo and ex vivo phagocytic potential of macrophages from progeny of breeder hens kept on ochratoxin A (OTA)-contaminated diet

This study aimed to investigate the phagocytic potential of macrophages in progeny of breeder hens kept on an OTA-contaminated diet. For this purpose, 84 White Leghorn (WL) layer breeder hens (40-weeks-of-age) were divided into seven groups (A–G). Hens in Group A were fed a commercial layer ration while those in Groups B–G were kept on a diet amended with 0.1, 0.5, 1.0, 3.0, 5.0, or 10.0?mg OTA/kg, respectively, for up to 3 weeks (n?=?12/treatment group; n?=?4/time sub-group/treatment group). Fertile eggs were set for hatching on a weekly basis to get the progeny of each week separately. Hatched chicks (n?=?10 from each group) were injected with India ink at day 14-of-age to study the in vivo phagocytosis of carbon particles. At day 30, abdominal macrophages were collected from 15 chicks/group and were used to assess their ex vivo/in vitro phagocytic potential against sheep red blood cells (SRBC) as well as for nitrite production upon challenge with lipopolysaccharide (LPS). The phagocytic indices of the reticuloendothelial system of all three sets of progeny (chicks obtained from hens fed OTA for 7, 14, and 21 days) were significantly lower than values seen with Group A chicks. The number of macrophages that were actively phagocytic, the number of SRBC internalized per macrophage, and the extent of nitrite production after stimulation with LPS were each significantly lower in the cells obtained from chicks of breeder hens that had been maintained on the OTA-contaminated diets. The findings of this study clearly showed that there are immunosuppressive effects—in terms of depressed in vivo and in vitro macrophage functionality—in progeny of OTA-fed breeder hens.     

Macrophage polarization following chitosan implantation

Macrophages are a key cell in the host response to implants and can be polarized into different phenotypes capable of inducing both detrimental and beneficial outcomes in tissue repair and remodeling, being important in tissue engineering and regenerative medicine. The objective of this study was to evaluate the macrophage response to 3D porous chitosan (Ch) scaffolds with different degrees of acetylation (DA, 5% and 15%). The M1/M2 phenotypic polarization profile of macrophages was investigated in vivo using a rodent air-pouch model. Our results show that the DA affects the macrophage response. Ch scaffolds with DA 5% induced the adhesion of lower numbers of inflammatory cells, being the M2 the predominant phenotypic profile among the adherent macrophages. In the inflammatory exudates F4/80⁺/CD206⁺ cells (M2 macrophages) appeared in higher numbers then F4/80⁺/CCR7⁺ cells (M1 macrophages), in addition, lower levels of pro-inflammatory cytokines together with higher levels of anti-inflammatory cytokines were found. Ch scaffolds with DA 15% showed opposite results, since M1 were the predominant macrophages both adherent to the scaffold and in the exudates, together with high levels of pro-inflammatory cytokines. In conclusion, Ch scaffolds with DA 5% induced a benign M2 anti-inflammatory macrophage response, whereas Ch scaffolds with DA 15% caused a macrophage M1 pro-inflammatory response.     

The influence of different culture microenvironments on the generation of dendritic cells from non-small-cell lung cancer patients

Introduction: Monocyte-derived dendritic cells (DCs) are currently under extensive evaluation as cell vaccines for cancer treatment. Many protocols regarding DCs generation in vitro with different protein components, especially autologous proteins, have been described. On the other hand, active tumor-derived factors in patients’ serum could impair monocytes, which might result in their abrogated differentiation into DCs in vitro. Materials and Methods: Autologous DCs from non-small-cell lung cancer (NSCLC)-bearing patients were generated in different culture microenvironments. Peripheral blood mononuclear cells (PBMCs) were cultured in the presence of interleukin-4 and granulocyte-monocyte-stimulating factor with supplementation of 10% autologous serum, 10% allogenic serum, or 2% human albumin. The course of apoptosis, phagocytic ability, and the immunophenotype of the generated DCs were analyzed using flow cytometric methods. Results: After 48 h of culture, we found a lower percentage of CD1a⁺/CD14⁺ and a higher percentage of CD1a⁺/CD14⁻ cells in the culture supplemented with human albumin than in the cultures supplemented with serums. The lowest CD14 antigen expression was found in the human albumin-supplemented 48-h cultures. After 48 h in the cultures carried out with human albumin we found significantly higher percentages of AV⁺/PI⁺ cells and AV⁻/PI⁺ cells than in cultures supplemented with autologous or allogenic serum. We also noted that the expression of FITC-dextran after 4 and 24 h of incubation was significantly higher in the cultures supplemented with both serums than in the HA-SC. The percentage of semi-mature DCs and of CD83 expression was lowest in the culture supplemented with 2% human albumin. Conclusions: The kind of culture supplementation had a great impact on the apoptosis of cultured PBMCs. It could also influence the yield of monocyte-derived DCs. It was also confirmed that autologous and allogenic serums provide suitable microenvironments for the generation of autologous DCs from NSCLC patients. The choice of culture supplementation for DC generation is still unsolved and further studies should be undertaken.