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PubMed2001

Tissue engineering: implications in the treatment of organ and tissue defects.

The loss or failure of organs and tissues due to injury/trauma or ageing is one of the major causes for concern in human health care. Tissue engineering, a new emerging field in modern biomedical sciences, promises a new ray of hope for transplantation biology by creating viable substitutes for such failing organs or tissues. Tissue engineering is rapidly growing in both scope and importance within biomedical engineering. It represents the marriage of rapid developments in cellular and molecular biology on the one hand and materials, chemical and mechanical engineering on the other. The ability to manipulate and reconstitute tissue function has tremendous clinical implications and is likely to play a key role in cell and gene therapies in the coming years. This article discusses the basic principles of tissue engineering and some of the attempts made to provide solutions by engineering tissues in vitro.

Aged↗

PubMed2003

A yeast display system for engineering functional peptide-MHC complexes.

In a cellular immune response, antigenic peptides derived by intracellular processing of foreign pathogens are bound to the class I major histocompatability complex (MHC I) and presented to CD8(+) cytotoxic T cells. Although the crystal structures of several different MHC products have been solved, many MHC molecules, including some associated with diseases, have not been amenable to biochemical and structural studies. The variability in this success is based largely on the fact that peptide-MHC complexes vary extensively in their stability. These properties also are intimately tied to the biological activity of the complexes. The ability to apply the techniques of directed evolution to this system in order to engineer stable complexes has been complicated by the trimeric structure of peptide-MHC complexes, requiring association of three polypeptides: the heavy chain, beta2-microglubulin (beta2m), and a short peptide. We show here that single-chain forms of peptide-MHC complexes can be expressed as Aga-2 fusions on the surface of yeast. Three different complexes, SIYRYYGL-K(b)-beta2m (SIYR-K(b)), EQYKFYSV-K(b)-beta2m (dEV8-K(b)), and SIINFEKL-K(b)-beta2m (OVA-K(b)), were expressed on yeast and detected by flow cytometry with a conformation-specific anti-K(b) antibody (B.8.24.3). In addition, yeast displaying K(b) loaded with exogenous SIYR and OVA peptides were recognized by a high-affinity T cell receptor that is specific for SIYR-K(b) and by an antibody (25.D1-16) that is specific for OVA-K(b), respectively. Finally, yeast that display the SIYRYYGL-K(b) also directly stimulated CD69 up-regulation on naive 2C T cells. Hence, yeast display represents a technology that can be used for directed evolution of any of the components of the trimeric pep-MHC complex.

Animals↗

PubMed2006

Monitoring of metabolite gradients in tissue-engineered constructs.

At present, the assessment of developing tissue-engineered constructs is almost always carried out destructively using biochemical or histological methods to determine cell number, viability and tissue growth throughout the construct. Since many of these experiments are long, taking weeks or even months to complete, simple and readily applicable non-destructive methods of monitoring changes in cell metabolism, viability and tissue deposition within the construct would be invaluable; such methods could point out adverse responses during the early stages of culture. Here, we describe the use of microdialysis for detecting local changes in cellular metabolism within a tissue-engineered construct. Three-dimensional constructs consisting of bovine articular chondrocytes entrapped in an alginate gel were cultured in a bioreactor for two weeks. Glucose and lactate were monitored by microdialysis, as the major nutrient and metabolite, respectively. Concentration gradients within the construct were evident, with the highest lactate concentrations in the construct centre. The local lactate concentration was a measure of cellular metabolic activity, decreasing as cellular activity fell and increasing as cellular activity was stimulated. Nutrient starvation and cell death in the construct centre could be readily detected in constructs deliberately cultured under adverse conditions. The results show that probe measurements can give an early warning of inappropriate local metabolic changes. Such information during the growth of tissue-engineered constructs would allow either corrective action or else an early end to an unsuccessful test.

Alginates↗

PubMed2005

The effect of structural alterations of PEG-fibrinogen hydrogel scaffolds on 3-D cellular morphology and cellular migration.

The need for alternative scaffolds in tissue engineering has motivated the establishment of advanced biomaterial technologies based on biosynthetic polymers. Networks of synthetic and biologic building blocks are created into a biomimetic environment for enhanced tissue compatibility with precise structural properties. The current investigation describes a unique biosynthetic hybrid scaffold comprised of synthetic polyethylene glycol (PEG) and endogenous fibrinogen precursor molecules. The PEGylated fibrinogen is cross-linked using photoinitation in the presence of cells to form a dense cellularized hydrogel network. The fibrin-like scaffold material maintains its biofunctionality through the fibrinogen backbone, while changes in the molecular architecture of the synthetic precursor are used to alter the nanostructrual properties of the scaffold, including mesh size and permeability. The structural properties of 6- and 10-kDa PEG-fibrinogen hydrogels are characterized by measuring the swelling properties and relating them to the degradation kinetics of the scaffold. Increased concentrations of the synthetic PEG are used to further alter the network structure of the PEG-fibrinogen hydrogel. Experiments using smooth muscle cells cultured inside the PEG-fibrinogen scaffold demonstrates a qualitative relationship between the molecular architecture of the matrix and the cellular morphology. A quantitative assessment of cell migration into the hydrogel network demonstrates a strong correlation between rate of cellular invasion and the network structure of the matrix. The ability to regulate cellular characteristics using structural modifications to the PEG-fibrinogen scaffold can be a valuable tool in tissue engineering and tissue regeneration.

Animals↗

PubMed2006

Three-dimensional nanohydroxyapatite/chitosan scaffolds as potential tissue engineered periodontal tissue.

The development of suitable three-dimensional scaffold for the maintenance of cellular viability and differentiation is critical for applications in periodontal tissue engineering. In this work, different ratios of porous nanohydroxyapatite/chitosan (HA/chitosan) scaffolds are prepared through a freeze-drying process. These scaffolds are evaluated in vitro by the analysis of microscopic structure, porosity, and cytocompatibility. The expression of type I collagen and alkaline phosphatase (ALP) activity are detected with real-time polymerase chain reaction (RT-PCR). Human periodontal ligament cells (HPLCs) transfected with enhanced green fluorescence protein (EGFP) are seeded onto the scaffolds, and then these scaffolds are implanted subcutaneously into athymic mice. The results indicated that the porosity and pore diameter of the HA/chitosan scaffolds are lower than those of pure chitosan scaffold. The HA/chitosan scaffold containing 1% HA exhibited better cytocompatibility than the pure chitosan scaffold. The expression of type I collagen and ALP are up-regulated in 1% HA/chitosan scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferate but also recruit surrounding tissue to grow in the scaffold. The degradation of the scaffold significantly decreased in the presence of HA. This study demonstrated the potential of HA/ chitosan scaffold as a good substrate candidate in periodontal tissue engineering.

Alkaline Phosphatase↗

PubMed1998

Materials for protein delivery in tissue engineering.

The ability of protein agents to modulate cellular behaviors, such as motility, proliferation, adhesion and function, is the subject of intense research; new therapies involving proteins will likely result. Unfortunately, many proteins have short half-lives and the potential for toxicity after systemic delivery, so traditional routes of administration are not appropriate. Alternate methods for sustained delivery of these agents to the desired cells and tissues in biologically active conformations and concentrations are necessary. Techniques similar to those long used in the controlled delivery of drugs have been used to administer certain growth factors to cells and tissues; although clinical success has been limited to date, studies in animal models suggest the potential for tremendous advances in the near future. This review outlines the basic technology of controlled protein delivery using polymeric materials, and discusses some of the techniques under investigation for the efficient administration of proteins in tissue engineering.

Journal Article↗

PubMed2004

Increased rate of chondrocyte aggregation in a wavy-walled bioreactor.

A novel wavy-walled bioreactor designed to enhance mixing at controlled shear stress levels was used to culture chondrocytes in suspension. Chondrocyte aggregation in suspensions mixed at 30, 50, and 80 rpm was characterized in the wavy-walled bioreactor and compared with that in conventional smooth-walled and baffled-walled spinner flask bioreactors. Aggregation was characterized in terms of the percentage of cells that aggregated over time, and aggregate size changes over time. The kinetics of chondrocyte aggregation observed in the bioreactors was composed of two phases: early aggregation between 0 and 2 h of culture, and late aggregation between 3 and 24 h of culture. At 50 rpm, the kinetics of early aggregation in the wavy-walled bioreactor was approximately 25% and 65% faster, respectively, than those in the smooth-walled and baffled-walled spinner flask bioreactors. During the late aggregation phase, the kinetics of aggregation in the wavy-walled bioreactor were approximately 45% and 65% faster, respectively, than in the smooth-walled and baffled-walled spinner flasks. The observed improved kinetics of chondrocyte aggregation was obtained at no cost to the cell survival rate. Results of computerized image analysis suggest that chondrocyte aggregation occurred initially by the formation of new aggregates via cell-cell interactions and later by the joining of small aggregates into larger cell clumps. Aggregates appeared to grow for only a couple of hours in culture before reaching a steady size, possibly determined by limitations imposed by the hydrodynamic environment. These results suggest that the novel geometry of the wavy-walled bioreactor generates a hydrodynamic environment distinct from those traditionally used to culture engineered cartilage. Such differences may be useful in studies aimed at distinguishing the effects of the hydrodynamic environment on tissue-engineered cartilage. Characterizing the wavy-walled bioreactor's hydrodynamic environment and its effects on cartilage cell/tissue culture can help establish direct relationships between hydrodynamic forces and engineered tissue properties.

Animals↗

PubMed2000

[Protection against influenza by genetically engineered vaccines].

Viral vector recombinant DNA vaccines, so called genetically engineered vaccines, are introduced directly into cells to induce an antigen specific immune response. This is a new approach to vaccination that represents a new concept in immunotherapy, which attempts to induce a long-term effective, anti-specific, viral antigen immune response. These vaccines may also prove more potent than currently used vaccines when it comes to inducing cellular immune responses in addition to humoral immune response. In the case of influenza, an immune response with humoral characteristics was induced using an influenza hemagglutinin(HA) DNA vaccine. On the other hand, injection of influenza virus nucleoprotein(NP) induced a Th-1 type cellular immune response including CTL. Genetically engineered vaccines have the potential to effectively overcome the problem of unresponsiveness to currently used vaccines.

Animals↗

PubMed2001

Cellular biosensors for drug discovery.

Recent advances in cell biology, fluorescent probe chemistry, miniaturization and automation have allowed the use of mammalian cells in a variety of medical and industrial applications. Here we describe the generation of cell-based biosensors, engineered to optically report specific biological activity. Cellular biosensors are comprised of living cells and can be used in various applications, including screening chemical libraries for drug discovery and environmental sensing. Panels of biosensors may also be useful for elucidating the function of novel genes. Here we describe two examples of the construction and use of engineered cell lines as biosensors for drug discovery.

Animals↗

PubMed2006

Enhancing the drug metabolism activities of C3A--a human hepatocyte cell line--by tissue engineering within alginate scaffolds.

In this study, we investigated the applicability of C3A--a human hepatocyte cell line--as a predicting tool for drug metabolism by applying tissue-engineering methods. Cultivation of C3A cells within alginate scaffolds induced the formation of spheroids with enhanced drug metabolism activities compared to that of two-dimensional (2-D) monolayer cultures. The spheroid formation process was demonstrated via histology, immunohistochemistry, and transmission electron microscope (TEM) analyses. The C3A spheroids displayed multilayer cell morphology, characterized by a large number of tight junctions, polar cells, and bile canaliculi, similar to spheroids of primary hepatocytes. Spheroid formation was accompanied by a reduction in P-glycoprotein (Pgp) gene expression and C3A cell proliferation was limited mainly to cells on the spheroid outskirt. The 3-D constructs maintained a nearly constant cell number according to MTT assay. Drug metabolism by the two most important cytochrome p-450 (CYP) enzymes in human liver, CYP1A2 and CYP3A4, was tested using preferred drugs. With CYP1A2, 3-fold enhancement in activity per cell was seen for converting ethoxyresorufin to resorufin compared to C3A cell monolayers. The spheroids responded to the inducer beta-naphthoflavone and to the inhibitor furafylline of CYP1A2. Enhanced metabolizing activity of CYP3A4, measured by the amount 6beta-testosterone formed from testosterone, and that of the phase II enzyme glucuronosyltransferases (UGT) further indicated that the tissue-engineered C3A spheroids may provide an efficient experimental tool for predicting drug activities by these CYPs. Moreover, the maintenance of constant cell number, as well as the elevated hepatocellular functions and drug metabolism activities, suggest that the tissue-engineered C3A may be applicable in replacement therapies.

Androgens↗

PubMed2000

Green fluorescent protein as a reporter for macromolecular localization in bacterial cells.

Green fluorescent protein (GFP) is a highly useful fluorescent tag for studying the localization, structure, and dynamics of macromolecules in living cells, and has quickly become a primary tool for analysis of DNA and protein localization in prokaryotes. Several properties of GFP make it an attractive and versatile reporter. It is fluorescent and soluble in a wide variety of species, can be monitored noninvasively by external illumination, and needs no external substrates. Localization of GFP fusion proteins can be analyzed in live bacteria, therefore eliminating potential fixation artifacts and enabling real-time monitoring of dynamics in situ. Such real-time studies have been facilitated by brighter, more soluble GFP variants. In addition, red-shifted GFPs that can be excited by blue light have lessened the problem of UV-induced toxicity and photobleaching. The self-contained domain structure of GFP reduces the chance of major perturbations to GFP fluorescence by fused proteins and, conversely, to the activities of the proteins to which it is fused. As a result, many proteins fused to GFP retain their activities. The stability of GFP also allows detection of its fluorescence in vitro during protein purification and in cells fixed for indirect immunofluorescence and other staining protocols. Finally, the different properties of GFP variants have given rise to several technological innovations in the study of cellular physiology that should prove useful for studies in live bacteria. These include fluorescence resonance energy transfer (FRET) for studying protein-protein interactions and specially engineered GFP constructs for direct determination of cellular ion fluxes.

Bacterial Proteins↗

PubMed2001

Reflections on the scope and the future of metabolic engineering and its connections to functional genomics and drug discovery.

Concepts, experience, and tools from metabolic engineering are immediately applicable to the challenge of understanding how the genome influences phenotype. However, new experimental approaches and mathematical and computational resources are needed to maximize the contributions of metabolic engineering to general questions in functional genomics. Among the priorities are systems for studying physiology on a microscale, theoretical tools for understanding biological control systems, and metabolic simulators "in silico" which provide reasonable predictions of stimulus-response relationships at engineering and medical resolution, with incomplete information on cellular mechanisms and their parameters. Approaching cells as complex systems, already a well-established principle in metabolic engineering, is essential to surmount stagnation in the rate of pharmaceutical discovery which is still based on a naive single-target paradigm.

Cells, Cultured↗

PubMed2006

Feasibility of vitrification as a storage method for tissue-engineered blood vessels.

It is well established that, in multicellular systems, conventional cryopreservation results in damaging ice formation, both in the cells and in the surrounding extracellular matrix. As an alternative to conventional cryopreservation, we performed a feasibility study using vitrification (ice-free cryopreservation) to cryopreserve tissue-engineered blood vessels. Fresh, frozen, and vitrified tissue-engineered blood vessels were compared using histological methods, cellular viability, and mechanical properties. Cryosubstitution methods were used to determine the location of ice in conventionally cryopreserved engineered vessels. Ice formation was negligible (0.0 +/- 0.0% of vessel area) in the vitrified specimens, and extensive (68.3 +/- 4.5% of vessel area) in the extracellular matrix of frozen specimens. The metabolic assay and TUNEL staining results indicated that vitrified tissue had similar viability to fresh controls. The contractility results for vitrified samples were >82.7% of fresh controls and, in marked contrast, the results for frozen samples were only 10.7% of fresh controls (p < 0.001). Passive mechanical testing revealed enhanced tissue strength after both freezing and vitrification. Vitrification is a feasible storage method for tissue-engineered blood vessel constructs, and their successful storage brings these constructs one step closer to clinical utility.

Animals↗

PubMed2004

Picolinic acid- or desferrioxamine-inducible autocrine activation of macrophages engineered to produce IFNgamma: an approach for gene therapy.

Macrophage (Mphi)-based vectors are highly mobile cellular shuttles designed to deliver therapeutic genes within the tissues. We engineered a mouse Mphi cell line to express the murine interferon-gamma (IFNgamma) under the control of an inducible promoter containing the hypoxia-responsive element, which can be triggered by hypoxia and other stimuli. We show that this Mphi vector can be induced to produce IFNgamma under normoxic conditions by stimulation with picolinic acid (PA), a catabolite of tryptophan, or desferrioxamine (DFX), an iron-chelating drug. The Mphi vector responds to IFNgamma with the induction of IRF-1 and of other IFNgamma-inducible genes, the expression of Ia antigens and induction of phagocytic activity. Inducible nitric oxygen synthase gene expression, nitric oxide production, as well as TNFalpha secretion were enhanced by PA or DFX as the sole stimuli. None of the above responses could be triggered individually by PA or DFX in control, normal Mphi, indicating that the Mphi vector overcame the need for costimulatory molecules derived from the immune system for its full activation. Furthermore, we demonstrate that extracellular iron can downregulate such response, thereby identifying an additional tool for the fine tuning of the Mphi vector response to stimulation.

Animals↗

PubMed2001

Engineering of macrophages to produce IFN-gamma in response to hypoxia.

Activation of murine macrophages (Mphi) requires the collaboration of signals derived from the immune system and the environment. In this study, we engineered a murine Mphi cell line to become activated in response to an environmental signal, hypoxia, as the sole stimulus. Hypoxia is a condition of low oxygen tension, occurring in several pathological tissues, which acts in synergy with IFN-gamma to induce full Mphi activation. We transfected the ANA-1 murine Mphi cell line with a construct containing the IFN-gamma gene controlled by a synthetic promoter inducible by hypoxia (HRE3x-Tk), and we characterized the cellular and molecular biology of the engineered Mphi under normoxia or hypoxia. Engineered Mphi in normoxia expressed basal levels of IFN-gamma mRNA and protein that were strongly augmented by shifting the cells to hypoxia. Furthermore, they responded to the synthesized IFN-gamma with induction of IFN-responsive factor-1 and 2'-5'-oligoadenylate synthase expression. Under normoxic conditions, the engineered Mphi had a significant constitutive level of Ia Ags and Fc receptors. Hypoxia induced further augmentation of Ia and Fc expression. Finally, hypoxia induced inducible NO synthase expression, and subsequent reoxygenation led to the production of NO. In conclusion, the engineered Mphi, which produce IFN-gamma in an inducible manner, express new biochemical and functional properties in response to low oxygen environment as the sole stimulus, thereby circumventing the need for costimulation by other immune system-derived signals.

Animals↗

PubMed2003

Normal features of tissue-engineered auricular cartilage by flow cytometry and histology: patient safety.

BACKGROUND: Cytokinetic abnormalities in DNA content, such as aneuploidy, haploidy, and tetraploidy, have been found to occur in human cartilaginous tumors. The high number of chondrocytes needed for tissue-engineered cartilaginous implants requires the cells to be passaged repeatedly. The theoretical risk of changes in the normal diploid state of these cells during their growth in vitro and after generation of tissue-engineered cartilage in vivo is not known. Materials and methods Auricular chondrocytes were obtained from 6 patients and cultured in vitro. Chondrocyte number was increased by repeated passaging. The passaged cells were implanted in nude mice for 8 weeks to generate tissue-engineered cartilage. Fresh control chondrocytes along with the passaged cells and cells obtained from the tissue-engineered constructs were collected and compared for DNA content by flow cytometry. RESULTS: Flow cytometry demonstrated 100% diploidy with no evidence of aneuploidy, haploidy, or tetraploidy in all groups of cells. Histology of the tissue-engineered cartilage also showed no evidence of cellular atypia. CONCLUSION: The number of human auricular chondrocytes can be increased by repeated passaging and passaged chondrocytes can be safely used for implantation to generate tissue-engineered constructs without a change in the normal diploid state of the cells. Histology of the cartilage generated showed normal features without atypia.

Aneuploidy↗

PubMed2001

Cellular integration of thyrocytes and thyroid folliculogenesis: a perspective for thyroid tissue regeneration and engineering.

Thyroid gland is composed of many spheroid structures called thyroid follicles, in which thyrocytes are integrated in their specific structural and functional polarization. In conventional monolayer and floating cultures, the cells cannot reorganize follicle structures with normal polarity. By contrast, in a 3-D collagen gel culture thyrocytes easily and stably reconstruct follicles with physiological polarity. Integration of thyrocyte growth and differentiation appears to result in eventual thyroid folliculogenesis. 3-D collagen gel culture and subacute thyroiditis, a specific thyroid disorder, are the promising models for addressing the mechanism of thyroid folliculogenesis. Because formation of 3-D follicles actively occurs both in this culture system and at the regenerative stage of the disease. The understanding of the mechanistic basis of folliculogenesis is prerequisite for establishment of an artificial thyroid tissue, which would enable a more physiological approach to the treatment of hypothyroidism caused by various diseases and surgical processes than conventional hormone replacement therapy. In this review, we have discussed thyrocyte integration, and thyroid folliculogenesis and tissue regeneration, to further thyroid biology. Also, we briefly discussed a perspective on thyroid tissue regeneration and engineering.

Animals↗

PubMed2005

Decellularization of bovine pericardium for tissue-engineering by targeted removal of xenoantigens.

BACKGROUND AND AIM OF STUDY: Assessment of decellularization of xenogeneic biological scaffolds for tissue engineering has relied primarily on histological cellularity, though this may not ensure the removal of known xenogeneic antigens such as galactose-alpha1,3-galactose (alpha-gal) and MHC I. METHODS: Bovine pericardium (BP) underwent standard (Std) decellularization consisting of hypotonic lysis and treatment with DNAse/RNAse. In addition to Std decellularization, tissues were treated for 24 h with either 0.5% Triton X-100, 0.5% sodium deoxycholate (SD), 0.1% sodium dodecyl sulfate (SDS), alpha-galactosidase (5 U/ml) or phospholipase (PL) A2 (150 U/ml). Tissues underwent a 96-h washout under gentle agitation at 27 degrees C, and then evaluated by light microscopy for % cellularity, and by immunohistochemistry and Western blot for alpha-gal, bovine MHC I and smooth muscle alpha-actin. RESULTS: Standard treatment of BP resulted in only partial removal histological cellularity and persistence of alpha-gal, MHC I and alpha-actin. Adding SD treatment resulted in apparent acellularity, but persistence of xenogeneic antigens. Only the addition of SDS resulted in complete histological acellularity and removal of xenogeneic antigens. Treatment with alpha-galactosidase selectively removed alpha-gal from BP. CONCLUSION: Histological cellularity is not an adequate end-point for assuring removal of antigenicity from xenogeneic biological scaffolds. However, known xenogeneic antigens can be targeted for removal by novel decellularization treatments such as alpha-galactosidase.

Actins↗