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PubMed2006

[Culture of chondrocytes using collagen-hydroxyapatite composite scaffolds in vitro].

OBJECTIVE: To evaluate the feasibility and the value of the layered cylindric collagen-hydroxyapatite composite as a scaffold for the cartilage tissue engineering after an observation of how it absorbs the chondrocytes and affects the cell behaviors. METHODS: The chondrocytes were isolated and multiplied in vitro, and then the chondrocytes were seeded onto the porous collagen/hydroxyapatite composite scaffold and were cultured in a three-dimensional environment for 3 weeks. The effects of the composite scaffold on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by the phase-contrast microscopy, histology, scanning electron microscopy, and immunohistochemistry. RESULTS: The pore diameter of the upper layer of the collagen-hydroxyapatite composite scaffold was about 147 microm, and the porosity was 89%; the pore diameter of the bottom layer was about 85 microm and the porosity was 85%. The layered cylindric collagen-hydroxyapatite composite scaffold had good hydrophilia. The chondrocytes that adhered to the surface of the scaffold, proliferated and migrated into the scaffold after 24 hours. The chondrocytes attached to the wall of the microholes of the scaffold maintained a rounded morphology and could secrete the extracellular matrix on the porous scaffold. CONCLUSION: The layered cylindric collagen-hydroxyapatite composite scaffold has a good cellular compatibility, and it is stronger in the mechanical property than the pure collagen. It will be an ideal scaffold for the cartilage tissue engineering.

Animals↗
PubMed2001

Modulation of cellular responses by plasmid CD40L: CD40L plasmid vectors enhance antigen-specific helper T cell type 1 CD4+ T cell-mediated protective immunity against herpes simplex virus type 2 in vivo.

Engineering gene therapy vectors to modulate the immune response is an important goal. In this regard, costimulation of T cells is a critical determinant in immune activation. The costimulatory molecule CD40, expressed on antigen-presenting cells, is thought to interact with CD40 ligand (CD40L) expressed on activated CD4(+) or CD8(+) T cells to further drive interleukin-2 receptor (IL-2R) expression and antigen-specific T cell expansion necessary for both class II and class I responses. To compare the specific roles of these two costimulatory molecules in immune induction in a herpes simplex virus (HSV) model, we constructed plasmid DNAs expressing CD40 and CD40L, coimmunized these molecules with a gD plasmid vaccine, and then analyzed immune modulatory effects as well as protection against lethal HSV-2 challenge. We observed that gD-specific IgG production was unaffected by these molecules. However, a higher production of IgG2a isotype was induced by CD40L coinjection, suggesting that CD40L drives immune responses towards a helper T cell type 1 (Th1) phenotype. CD40L also enhanced Th cell proliferative responses and production of Th1-type cytokines (IL-2 and IFN-gamma) and beta-chemokines (RANTES and MIP-1alpha) from splenocytes. In contrast, CD40 showed slightly increasing effects on T cell proliferation responses and cytokine and chemokine production. When animals were challenged with a lethal dose of HSV-2, CD40L-coimmunized animals exhibited a significantly enhanced survival rate, as compared with CD40 coinjection or gD DNA vaccine alone. This enhanced protection appears to be mediated by Th1-type CD4(+) T cells, as determined by in vitro and in vivo T cell subset deletion. CD40L also promoted migration of CD4(+) T cells into the muscle sites. These studies demonstrate that CD40L can play an important role in protective antigen-specific immunity in a gene-based model system through increased expansion of the CD4(+) Th1 T cell subset in vivo.

Adjuvants, Immunologic↗
PubMed1998

The optimization of helper T lymphocyte (HTL) function in vaccine development.

Helper T lymphocyte (HTL) responses play an important role in the induction of both humoral and cellular immune responses. Therefore, HTL epitopes are likely to be a crucial component of prophylactic and immunotherapeutic vaccines. For this reason, Pan DR helper T cell epitopes (PADRE), engineered to bind most common HLA-DR molecules with high affinity and act as powerful immunogens, were developed. Short linear peptide constructs comprising PADRE and Plasmodium-derived B cell epitopes induced antibody responses comparable to more complex multiple antigen peptides (MAP) constructs in mice. These antibody responses were composed mostly of the IgG subclass, reactive against intact sporozoites, inhibitory of schizont formation in liver invasion assays, and protective against sporozoite challenge in vivo. The PADRE HTL epitope has also been shown to augment the potency of vaccines designed to stimulate a cellular immune response. Using a HBV transgenic murine model, it was found that CTL tolerance was broken by PADRE-CTL epitope lipopeptide, but not by a similar construct containing a conventional HTL epitope. There are a number of prophylactic vaccines that are of limited efficacy, require multiple boosts, and/or confer protection to only a fraction of the immunized population. Also, in the case of virally infected or cancerous cells, new immunotherapeutic vaccines that induce strong cellular immune responses are desirable. Therefore, optimization of HTL function by use of synthetic epitopes such as PADRE or pathogen-derived, broadly crossreactive epitopes holds promise for a new generation of highly efficacious vaccines.

Animals↗
PubMed2003

Comparison of multi-lineage cells from human adipose tissue and bone marrow.

Our laboratory has recently characterized a population of cells from adipose tissue, termed processed lipoaspirate (PLA) cells, which have multi-lineage potential similar to bone-marrow-derived mesenchymal stem cells (MSCs). This study is the first comparison of PLA cells and MSCs isolated from the same patient. No significant differences were observed for yield of adherent stromal cells, growth kinetics, cell senescence, multi-lineage differentiation capacity, and gene transduction efficiency. Adipose tissue is an abundant and easily procured source of PLA cells, which have a potential like MSCs for use in tissue-engineering applications and as gene delivery vehicles.

Adipose Tissue↗
PubMed2001

Radiosensitivity of mammalian cell lines engineered to overexpress cytosolic glutathione peroxidase.

Reactive oxygen species are believed to be involved in radiation lethality. Glutathione peroxidase is an intracellular enzyme with antioxidant functions. To determine whether increasing the cellular antioxidant capacity can confer radiation resistance, the effect of overexpression of glutathione peroxidase on radiosensitivity was determined in two different cell types. An expression construct including the bovine cytosolic glutathione peroxidase cDNA was used to overexpress this enzyme in cells of the human lymphoblast cell line Sup-T1 as well as the Chinese hamster ovary cell line AA8. Supplementation of the culture media with 30 nM sodium selenite was included to obtain optimal glutathione peroxidase activity. Northern blot analysis confirmed the presence of the construct mRNA, and a standard coupled spectrophotometric assay demonstrated significantly increased glutathione peroxidase activity in the transfected cell lines. An approximately 8-fold increase was found in the Sup-T1 cells, and an approximately 30-fold increase was obtained in the Chinese hamster ovary AA8 cells. Clonogenic survival was assayed in the overexpressing cells and compared to that in control cells transfected with vector alone. Despite significantly increased glutathione peroxidase activity, no observable radioprotection was conferred in either of the two cell lines studied, indicating that increased glutathione peroxidase activity is insufficient to confer radioresistance in the two cell types examined. These data are discussed in the context of using antioxidants as adjuncts to clinical radiotherapy.

Animals↗
PubMed2005

Molecular, cellular and developmental biology of urothelium as a basis of bladder regeneration.

Urinary bladder malfunction and disorders are caused by congenital diseases, trauma, inflammation, radiation, and nerve injuries. Loss of normal bladder function results in urinary tract infection, incontinence, renal failure, and end-stage renal dysfunction. In severe cases, bladder augmentation is required using segments of the gastrointestinal tract. However, use of gastrointestinal mucosa can result in complications such as electrolyte imbalance, stone formation, urinary tract infection, mucous production, and malignancy. Recent tissue engineering techniques use acellular grafts, cultured cells combined with biodegradable scaffolds, and cell sheets. These techniques are not all currently applicable for human bladder reconstruction. However, new avenues for bladder reconstruction maybe facilitated by a better understanding of urogenital development, the cellular and molecular biology of urothelium, and cell-cell interactions, which modulate tissue repair, homeostasis, and disease progression.

Animals↗
PubMed2004

Cationic polyelectrolyte hydrogel fosters fibroblast spreading, proliferation, and extracellular matrix production: Implications for tissue engineering.

Fibrous encapsulation is known to occur to many prosthetic implants and is thought to be due to the cells not adhering adequately to the surface. For developing new materials able to enhance cellular adhesion by mimicking extracellular matrix components, polyelectrolyte polymers, characterized by tunable surface charges, have been proposed. Here we demonstrate that panoply of cell functions over a two-dimensional substratum is influenced by surface charge. We have at first generated structurally related polyelectrolyte substrata varying in their positive surface charge amount and subsequently evaluated a variety of behaviors of human primary fibroblasts seeded on these polymers. The proportion of adherent, spreading, and proliferating cells was increased significantly on cationic hydrophilic surfaces when compared with the neutral base surface. The extent of cell spreading correlated with cytoskeleton organization as assessed using immunofluorescence techniques. In the key experiment, the presence of cationic charges on cell adhesion-resistant neutral surface increased the synthesis of collagen I and III, the release of their metabolites, and the expression of their mRNA by fibroblasts. Interestingly, the scarce collagen deposits on neutral polymer consisted, for the most part, of collagen I while collagen III was present only in traces probably due to the secretion of metalloproteinase-2 by non-adherent fibroblasts. Taken together, these results show that polyelectrolyte films may promote the attachment of fibroblast cells as well as their normal secretory phenotype. Both effects could be potentially useful in integrating soft connective tissue to the implant, decreasing the chance of its fibrous encapsulation.

Adult↗
PubMed2002

Evaluation of the biocompatibility of a chitosan scaffold in mice.

Chitosan scaffolds appear to be suitable for a variety of tissue engineering applications. This study addressed the biocompatibility of chitosan in a mouse implantation model. Porous chitosan scaffolds were implanted in mice, and animals were sacrificed after 1, 2, 4, 8, or 12 weeks. Macroscopic inspection of the implantation site revealed no pathological inflammatory responses. Histological assessment indicated marked neutrophil accumulation within the implant, which resolved with increasing implantation time. Gram staining and limulus assays revealed no evidence of infection or endotoxin. Collagen was observed within the chitosan pore spaces, indicating that connective tissue matrix was deposited within the implant. Angiogenic activity associated with the external implant surface was also observed. Cellular immune responses were determined by lymphocyte proliferation assays, and antibody responses were measured using ELISA techniques. These assays indicated a very low incidence of chitosan-specific reactions. Although there was a large migration of neutrophils into the implantation area, there were minimal signs of any inflammatory reaction to the material itself. This preliminary study demonstrates that chitosan has a high degree of biocompatibility in this animal model. Overall, the findings suggest that chitosan may be suitable for the development of implantable materials.

Animals↗
PubMedDate not supplied

Electrospun nanofiber fabrication as synthetic extracellular matrix and its potential for vascular tissue engineering.

Substantial effort is being invested by the bioengineering community to develop biodegradable polymer scaffolds suitable for tissue-engineering applications. An ideal scaffold should mimic the structural and purposeful profile of materials found in the natural extracellular matrix (ECM) architecture. To accomplish this goal, poly (L-lactide-co-epsilon-caprolactone) [P(LLA-CL)] (75:25) copolymer with a novel architecture produced by an electrospinning process has been developed for tissue-engineering applications. The diameter of this electrospun P(LLA-CL) fiber ranges from 400 to 800 nm, which mimicks the nanoscale dimension of native ECM. The mechanical properties of this structure are comparable to those of human coronary artery. To evaluate the feasibility of using this nanofibrous scaffold as a synthetic extracellular matrix for culturing human smooth muscle cells and endothelial cells, these two types of cells were seeded on the scaffold for 7 days. The data from scanning electron microscopy, immunohistochemical examination, laser scanning confocal microscopy, and a cell proliferation assay suggested that this electrospun nanofibrous scaffold is capable of supporting cell attachment and proliferation. Smooth muscle cells and endothelial cells seeded on this scaffold tend to maintain their phenotypic shape. They were also found to integrate with the nanofibers to form a three-dimensional cellular network. These results indicate a favorable interaction between this synthetic nanofibrous scaffold with the two types of cells and suggest its potential application in tissue engineering a blood vessel substitute.

Biocompatible Materials↗
PubMed1999

IL-12 delivery from recombinant vaccinia virus attenuates the vector and enhances the cellular immune response against HIV-1 Env in a dose-dependent manner.

To develop vaccination strategies against HIV-1 infection aimed to specifically enhance the cell-mediated immunity (CMI), we have engineered vaccinia virus (VV) recombinants expressing HIV-1 Env (rVVenv) and murine IL-12 (rVVlucIL-12) genes or coexpressing both genes (rVVenvIL-12). In mice inoculated with rVVlucIL-12 there is a rapid clearance of the virus, and this correlates with the induction of high levels of IL-12 and IFN-gamma in serum and spleen early after infection. Enzyme-linked immunospot analysis of mice inoculated with rVVlucIL-12, revealed a nearly 2-fold increase in the number of specific anti-VV CD8+ T cells compared with that in mice given control rVV, and the serum Ab response was biased in favor of a Th1 response. An enhancement of about 2-fold in the number of anti-gp160 IFN-gamma-secreting CD8+ T cells was observed in mice inoculated with rVVenvIL-12, when a dose of 1 x 107 PFU/mouse was used, but this enhancement was not observed when mice were given 5 x 107 PFU. This variation with virus dosage was confirmed in mice immunized simultaneously with different multiplicities of rVV expressing singly the env or IL-12 genes. The highest specific CMI was obtained in mice coadministered a low dose (2 x 104 PFU) of rVVlucIL-12 and 1 x 107 PFU of rVVenv. Our findings provide evidence for specific enhancement of the CMI to HIV-1 Env by the differential expression of IL-12 and env genes delivered from VV recombinants. This approach can be of wide vaccination interest as a means to improve immune responses to other Ags.

Adjuvants, Immunologic↗
PubMed1996

Site-directed mutagenesis enabled preparation of a functional fluorescent analog of profilin: biochemical characterization and localization in living cells.

The preparation of fluorescent profilin analogs for binding and spectroscopic studies, in vitro and in vivo, has been hampered by the poor chemical reactivity of this protein in its native form. We have addressed this problem by labeling a mutant, chemically reactive form of profilin. Site-directed mutagenesis was first used to replace a serine residue in a non-essential domain with a reactive cysteine residue. The mutant protein was expressed in Escherichia coli and reacted with tetramethylrhodamine iodoacetamide. In vitro assays indicated that the fluorescent profilin maintained its ability to bind actin, polyproline, and PIP2, to inhibit actin polymerization, and to stimulate actin nucleotide exchange. Fluorescence spectroscopy showed that neither the excitation nor the emission of the analog was sensitive to the interaction with actin or polyproline. However, binding of PIP2 caused a 75% quenching of the fluorescent signal, suggesting a dramatic change in the immediate environment of the probe. When the fluorescent profilin was microinjected into living NRK cells, it became localized at cell-cell junctions and discrete sites near the anterior end, where it colocalized with aggregates of unpolymerized actin. Different engineered forms of profilin with fluorophores located at defined sites should greatly facilitate the study of its interactions with various ligands and cellular structures.

Animals↗
PubMed2003

Insights into the relation between mRNA and protein expression patterns: I. Theoretical considerations.

Translation is a central cellular process in every organism and understanding translation from the systems (genome-wide) perspective is very important for medical and biochemical engineering applications. Moreover, recent advances in cell-wide monitoring tools for both mRNA and protein levels have necessitated the development of such a model to identify parameters and conditions that influence the mapping between mRNA and protein expression. Experimental studies show a lack of correspondence between mRNA and protein expression profiles. In this study, we describe a mechanistic genome-wide model for translation that provides mapping between changes in mRNA levels and changes in protein levels. We use our model to study the system in detail and identify the key parameters that affect this mapping. Our results show that the correlation between mRNA and protein levels is a function of both the kinetic parameters and concentration of ribosomes at the reference state. In particular, changes in concentration of free and total ribosomes in response to a perturbation; changes in initiation and elongation kinetics due to competition for aminoacyl tRNAs; changes in termination kinetics; average changes in mRNA levels in response to the perturbation; and changes in protein stability are all important determinants of the mapping between mRNA and protein expression.

Bacteria↗
PubMed2003

Myosin heavy chain kinase B participates in the regulation of myosin assembly into the cytoskeleton.

Myosin II plays critical roles in events such as cytokinesis, chemotactic migration, and morphological changes during multicellular development. The amoeba Dictyostelium discoideum provides a simple system for the study of this contractile protein. In this system, myosin II filament assembly is regulated by myosin heavy chain (MHC) phosphorylation in the tail region of the molecule. Earlier studies identified an alpha-kinase, MHC kinase A (MHCK A), which phosphorylates three mapped threonine residues in the myosin tail, driving myosin disassembly. Using molecular and genomic approaches, we have identified a series of related kinases in Dictyostelium. The enzyme MHCK B shares with MHCK A a domain organization that includes a highly novel catalytic domain coupled to a carboxyl-terminal WD repeat domain. We have engineered, expressed, and purified a FLAG-tagged version of the novel kinase. In the present study, we report detailed biochemical and cellular studies documenting that MHCK B plays a physiological role in the control of Dictyostelium myosin II assembly and disassembly during the vegetative life of Dictyostelium amoebae. The presented data supports a model of multiple related MHCKs in this system, with different regulatory mechanisms and pathways controlling each enzyme.

Animals↗
PubMed2001

Innovations in generation and analysis of 2D [(13)C,(1)H] COSY NMR spectra for metabolic flux analysis purposes.

2D [(13)C,(1)H] COSY NMR is used by the metabolic engineering community for determining (13)C-(13)C connectivities in intracellular compounds that contain information regarding the steady-state fluxes in cellular metabolism. This paper proposes innovations in the generation and analysis of these specific NMR spectra. These include a computer tool that allows accurate determination of the relative peak areas and their complete covariance matrices even in very complex spectra. Additionally, a method is introduced for correcting the results for isotopic non-steady-state conditions. The proposed methods were applied to measured 2D [(13)C,(1)H] COSY NMR spectra. Peak intensities in a one-dimensional section of the spectrum are frequently not representative for relative peak volumes in the two-dimensional spectrum. It is shown that for some spectra a significant amount of additional information can be gained from long-range (13)C-(13)C scalar couplings in 2D [(13)C,(1)H] COSY NMR spectra. Finally, the NMR resolution enhancement by dissolving amino acid derivatives in a nonpolar solvent is demonstrated.

Amino Acids↗
PubMed2000

Endothelial cells in culture: a model for studying vascular functions.

Vascular endothelium - lining the inner side of blood bessels - is one of the largest secretory tissues of the body. Therefore, understanding the cellular and molecular biology of the endothelial cells is essential for the development of new approaches for both the prevention and therapy of cardiovascular diseases. To this aim, in vitro cultures of endothelial cells provide a valuable technical resource. This review focuses on some of the critical phases of the endothelial cells culturing methodology such as: i) isolation and growing of endothelial cells; ii) identification of endothelial cells by morphological, biochemical and cellular markers; iii) studying endothelial cells in function of vascular pharmacology, vasomotor tone, vessel remodelling (angiogenesis/apoptosis), blood haemostasis, inflammatory reactions, and molecular engineering. Practical suggestions for culturing endothelial cells are presented while pros and cons of each method are discussed.

Animals↗
PubMed2006

Endothelialization and flow conditioning of fibrin-based media-equivalents.

It is generally accepted that endothelialization and subsequent development of a functional endothelium are of paramount importance to the success of any bioartificial artery. In this study, we aimed to assess the ability of smooth muscle cell-remodeled, fibrin-based media-equivalents (MEs) to be endothelialized, examine the morphological changes of endothelial cells (ECs) associated with exposure to physiologically-relevant shear stress in a custom-built bioreactor, and determine if adherent ECs are capable of withstanding average physiological shear stresses. It was found that MEs could be readily endothelialized with surface coverages of 98.8 +/- 0.9% after two days, and the ECs expressed von Willebrand factor. Furthermore, EC retention remained high (steady: 96.5 +/- 4.4%, pulsatile: 94.3 +/- 4.3%) under exposure to physiologically relevant shear stresses for 48 h. The results indicate that these MEs are conducive to generating an EC monolayer, with the ECs possessing adhesion strength sufficient to withstand physiological shear stress and maintain a normal phenotype.

Adaptation, Physiological↗
PubMed2006

Genetic algorithms as a tool for helix design--computational and experimental studies on prion protein helix 1.

Evolutionary computing is a general optimization mechanism successfully implemented for a variety of numeric problems in a variety of fields, including structural biology. We here present an evolutionary approach to optimize helix stability in peptides and proteins employing the AGADIR energy function for helix stability as scoring function. With the ability to apply masks determining positions, which are to remain constant or fixed to a certain class of amino acids, our algorithm is capable of developing stable helical scaffolds containing a wide variety of structural and functional amino acid patterns. The algorithm showed good convergence behaviour in all tested cases and can be parameterized in a wide variety of ways. We have applied our algorithm for the optimization of the stability of prion protein helix 1, a structural element of the prion protein which is thought to play a crucial role in the conformational transition from the cellular to the pathogenic form of the prion protein, and which therefore poses an interesting target for pharmacological as well as genetic engineering approaches to counter the as of yet uncurable prion diseases. NMR spectroscopic investigations of selected stabilizing and destabilizing mutations found by our algorithm could demonstrate its ability to create stabilized variants of secondary structure elements.

Algorithms↗
PubMed2004

Influence of nanoporous alumina membranes on long-term osteoblast response.

A major goal of bone tissue engineering is to design better scaffold configuration and materials to better control osteoblast behavior. Nanoporous architecture has been shown to significantly affect cellular response. In this work, nanoporous alumina membranes were fabricated by a two-step anodization method to investigate bone cell response. Osteoblasts were seeded on nanoporous alumina membranes to investigate both short-term adhesion and proliferation and long-term functionality and matrix production. Cell adhesion and proliferation were characterized using a standard MTT assay and cell counting. The total protein content was measured after cell lysis using the BCA assay. Matrix production was characterized in terms of surface concentrations of calcium and phosphorous, components of bone matrix, using X-ray photoelectron spectroscopy (XPS). The results from nanoporous alumina membranes were compared with those of amorphous alumina, aluminum, commercially available ANOPORE and glass. Results indicate improved osteoblast adhesion and proliferation and increased matrix production after 4 weeks of study.

Aluminum↗