Article

The Potential of Bioartificial Tissues in Oncology Research and Treatment

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

This review article addresses the relevance and potential of bioartificial tissues in oncologic research and therapy and reconstructive oncologic surgery. In order to translate the findings from basic cellular research into clinical applications, cell-based models need to recapitulate both the 3D organization and multicellular complexity of an organ but at the same time accommodate systematic experimental intervention. Here, tissue engineering, the generation of human tissues and organs in vitro, provides new perspectives for basic and applied research by offering 3D tissue cultures resolving fundamental obstacles encountered in currently applied 2D and 3D cell culture systems. Tissue engineering has already been applied to create replacement structures for reconstructive surgery. Applied in vitro, these complex multicellular 3D tissue cultures mimic the microenvironment of human tissues. In contrast to the currently available cell culture systems providing only limited insight into the complex interactions in tissue differentiation, carcinogenesis, angiogenesis and the stromal reaction, the more realistic (micro)environment afforded by the bioartificial tissuespecific 3D test systems may accelerate the progress in design and development of cancer therapies.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... More recently, this promising technology has been applied to the field of oncology with some attempts to develop engineered tumor tissues for pre-clinical research (e.g. human melanoma model) [78]. Here we took advantage of our tissue engineering know-how in the respiratory field [79] to develop a complex, but accessible, 3D lung cancer model: OncoCilAir™ [80,81]. ...
Chapter
Full-text available
In the continuity of the pioneering work of Donald Ingber (organ on chip), a series of 3D lung-on-a-chip microfluidic devices have been developed. Briefly, lung-on-a-chip is a biomimetic microsystem that reconstitutes the critical functional alveolar-capillary interface of the human lung, with periodic mechanical stretching and flow of the medium carrying immune cells. Using this micro-fluidic device, the authors were able to replicating the immune responses against bacterial infections in vitro [69]. Afterwards, devices were optimized as a drug screening platform to select individualized treatment for lung cancer. In these systems, lung cancer and stromal cell lines were co-cultured as 3D spheroids under continuous media supplementation, mimicking the circulation of nutrients and metabolic waste out of the cultures [70]. Another similar model has been developed for chemoresistive testing of pleural mesothelioma cancer spheroids. Interestingly, growth inhibitory concentration of cisplatin showed higher concentration in perfused tumor spheroids compared with spheroids cultured under static conditions [71]. These systems represent therefore valuable tools to get information about the efficacy of chemotherapeutic drugs in a dynamic microenvironment which recapitulate the actual in vivo situation, but they do not address side-toxicity on normal lung physiology. The challenge will be to improve the model so that it incorporates normal and functional tissues. That could be achieved by connecting such devices with other microphysiological organotypic chips, representative of healthy lung tissues.
... The nutrient becomes scarce rapidly due to the limited number of normal tissue vasculature present in the tissue. This leads to the tumour being incapable of acquiring sufficient nutrients, oxygen and tabolites to support continued exponential growth [3]. It has been found that tumour cells produce smaller amounts of diffusible inhibitory substances, which can limit cells proliferation and viability [4], [5]. ...
Conference Paper
Full-text available
The aim of this paper is to present a two-dimensional simulation tool based on several mathematical models, visualizing the growth and development of a solid tumour in avascular growth phase, vascular growth phase and the progress of angiogenesis. The avascular tumour remains in a diffusion-limited state of a few millimeters in diameter, while during vascularized phase, tumour cell proliferating rapidly and being able to invade the surrounding host tissue and blood system. Tumour-induced angiogenesis, the growth of a capillary network of blood vessels from a pre-existing vasculature, provides the crucial link between the two stages. Under different molecular mechanisms, this discrete model demonstrates several important factors affecting tumour growth at each stage. The simulation results are presented in two-dimensional combined with quantitatively surveyed. The long-term goal of this work is to develop a multicellular engine for precisely simulating and visualizing the insight into cancer biology.
... Until now, only human skin organ models have been established that are built of dermal fibroblasts – embedded in a bio-matrix from tissue-typical matrix proteins as feeder layer – and epidermal keratinocytes. This basic model is very flexible and it is used to study penetration, irritation and biocompatibility, as well as for wound healing and infection (Walles et al., 2007). However, fish skin models for pharmacological or analytical purposes are not established yet. ...
Article
Here, we report the establishment of a permanent skin cell culture from rainbow trout (Oncorhynchus mykiss). The cells of the fish skin cell culture could be propagated over 60 passages so far. Furthermore, we show for the first time that it is possible to integrate freshly harvested rainbow trout scales into this new fish skin cell culture. We further demonstrated that epithelial cells derived from the scales survived in the artificial micro-environment of surrounding fibroblast-like cells. Also, antibody staining indicated that both cell types proliferated and started to build connections with the other cell type. It seems that it is possible to generate an 'artificial skin' with two different cell types. This could lead to the development of a three-dimensional test system, which might be a better in vitro representative of fish skin in vivo than individual skin cell lines.
Thesis
Full-text available
Da in den letzten Jahrzehnten nur geringfügige Verbesserungen der Überlebensraten bei an einem Pankreaskarzinom erkrankten Patienten erzielt wurden, besteht ein dringender klinischer Bedarf für die Entwicklung wirksamer therapeutischer Strategien. Dreidimensionale in vitro Modelle sind für das Screening und die Validierung von Therapeutika essenziell. In der vorliegenden Arbeit konnte mittels der Methoden des Tissue Engineerings ein biolumineszenzbasiertes dreidimensionales in vitro Testsystem des pankreatischen Karzinoms aufgebaut und charakterisiert werden. Für die Detektion von LumineszenzIntensitäten wurde die pankreatische Krebszelllinie PANC-1 zuvor mit firefly luciferase (FLUC) transduziert. PANC-1 FLUC Zellen wurden auf porziner Pankreasmatrix (PanMa) und Dünndarmmatrix (SISser) kultiviert, um den Einfluss unterschiedlicher Matrizen auf das Verhalten der Zellen im Tumormodell zu untersuchen. Darüber hinaus wurden in dieser Arbeit die PANC-1 FLUC mit einem Standardtherapeutikum der Pankreaskarzinomtherapie, Gemcitabin, behandelt und die Wirkung mittels biolumineszenbasierter Bildgebung detektiert. Es konnte gezeigt werden, dass die Lumineszenz-Intensität von PANC-1 FLUC Zellen einer bestimmten Zellzahl durch biolumineszenzbasierte Messverfahren zugeordnet werden kann. Weiter wurde nachgewiesen, dass die Extrazellulärmatrix einen Einfluss auf die Expression tumorspezifischer Marker hat und PANC-1 FLUC Zellen ein unterschiedlich invasives Wachstum auf organspezifischen Matrizen aufweisen. Die Wirkung von Gemcitabin auf die Tumorzellen kann durch das hier vorgestellte biolumineszenzbasierte Messverfahren detektiert werden. Die in dieser Arbeit vorgestellten Ergebnisse sind die Grundlage für die weitere Validierung eines biolumineszenzbasierten dreidimemsionalen in vitro Testystems des pankreatischen Karzinoms für die präklinische Erforschung neuartiger Therapiestrategien.
Article
Solid tumors impose immunological and physical barriers to the efficacy of chimeric antigen receptor (CAR) T-cell therapy that are not reflected in conventional pre-clinical testing against singularized tumor cells in two-dimensional culture. Here, we established microphysiologic three-dimensional (3D) lung and breast cancer models that resemble architectural and phenotypical features of primary tumors, and evaluated the anti-tumor function of ROR1-specific CAR T-cells. 3D tumors were established from A549 (non-small cell lung cancer) and MDA-MB-231 (triple-negative breast cancer) cell lines on a biological scaffold with intact basement membrane (BM) under static and dynamic culture conditions, which resulted in progressively increasing cell mass and invasive growth phenotype (dynamic>static; MDA-MB-231>A549). Treatment with ROR1-CAR T-cells conferred potent anti-tumor effects. In dynamic culture, CAR T-cells actively entered arterial medium flow, adhered to and infiltrated the tumor mass. ROR1-CAR T-cells penetrated deep into tumor tissue and eliminated multiple layers of tumor cells located above and below the BM. The microphysiologic 3D tumor models developed in this study are standardized scalable test systems that can be used either in conjunction with or in lieu of animal testing to interrogate the anti-tumor function of CAR T-cells, and to obtain proof-of-concept for their safety and efficacy prior to clinical application.
Article
We developed methods to generate a biological vascularised scaffold [1] and induce angiogenesis in vitro [2]. This vascularised matrix enables the generation of a functional artificial vascular network. Standardization of co-cultures on this vascularized biological matrix should overcome the problem of lacking graft vascularisation and permitting the generation of complex bioartificial organ-like tissues in vitro. This model offers the possibility to study in vitro angiogenesis of human endothelial cells. The generated bioartificial tissues serve as test systems for pharmaceutical drug screening and patient specific therapy [3]. Target screening requires testsystems that mimic the human tissues with increasing accuracy in order to optimize the selection of potential effectors. Our vascularised bioartificial tissues offers the first time the simulation of physiological drug application, studies regarding biotransformation and toxic metabolites, and the establishment nanomaterials/drug delivery systems for research and therapy. First clinical applications addressed fundamental issues regarding ECM remodelling, functionality and thrombogenity of the vascular network [4].
Article
One of the most technical difficulties in tissue engineering (TE) is the development of conditions that direct cell behavior physiologically, and recapitulation of these conditions in a suitable in vitro growth environment. Scaffolds have been developed for numerous TE applications with initial success in tissues of relatively simple architecture, including skin and cartilage. Whereas the generation of tissues with greater complexity and the development of functional vascular networks in complex bioartificial tissues has been rather unsuccessful. The extracellular matrix and its tissue specific components in vivo demonstrate the role of bioactive molecules and structures in regulating cell behavior and tissue function. The majority of in vitro functional complex artificial tissue constructs and transplants are based on decellularized donor tissues. These acellular biological scaffolds are procuring a biomimetic microenvironment; an ideal cellular surrounding even for the in vitro angiogenesis. These biomimetic scaffolds may serve as blueprints for synthetical biological scaffolds including adhesive and physical factors to regulate cell adhesion, proliferation, tissue architecture, and function.
Article
Full-text available
For the development of new treatment strategies against cancer, understanding signaling networks and their changes upon drug response is a promising approach to identify new drug targets and biomarker profiles. Pre-requisites are tumor models with multiple read-out options that accurately reflect the clinical situation. Tissue engineering technologies offer the integration of components of the tumor microenvironment which are known to impair drug response of cancer cells. We established three-dimensional (3D) lung carcinoma models on a decellularized tissue matrix, providing a complex microenvironment for cell growth. For model generation, we used two cell lines with (HCC827) or without (A549) an activating mutation of the epidermal growth factor receptor (EGFR), exhibiting different sensitivities to the EGFR inhibitor gefitinib. EGFR activation in HCC827 was inhibited by gefitinib, resulting in a significant reduction of proliferation (Ki-67 proliferation index) and in the induction of apoptosis (TUNEL staining, M30-ELISA). No significant effect was observed in conventional cell culture. Results from the 3D model correlated with the results of an in silico model that integrates the EGFR signaling network according to clinical data. The application of TGFβ1 induced tumor cell invasion, accompanied by epithelial-mesenchymal transition (EMT) both in vitro and in silico. This was confirmed in the 3D model by acquisition of mesenchymal cell morphology and modified expression of fibronectin, E-cadherin, β-catenin and mucin-1. Quantitative read-outs for proliferation, apoptosis and invasion were established in the complex 3D tumor model. The combined in vitro and in silico model represents a powerful tool for systems analysis.
Article
Full-text available
Despite major scientific, medical and technological advances over the last few decades, a cure for cancer remains elusive. The disease initiation is complex, and including initiation and avascular growth, onset of hypoxia and acidosis due to accumulation of cells beyond normal physiological conditions, inducement of angiogenesis from the surrounding vasculature, tumour vascularization and further growth, and invasion of surrounding tissue and metastasis. Although the focus historically has been to study these events through experimental and clinical observations, mathematical modelling and simulation that enable analysis at multiple time and spatial scales have also complemented these efforts. Here, we provide an overview of this multiscale modelling focusing on the growth phase of tumours and bypassing the initial stage of tumourigenesis. While we briefly review discrete modelling, our focus is on the continuum approach. We limit the scope further by considering models of tumour progression that do not distinguish tumour cells by their age. We also do not consider immune system interactions nor do we describe models of therapy. We do discuss hybrid-modelling frameworks, where the tumour tissue is modelled using both discrete (cell-scale) and continuum (tumour-scale) elements, thus connecting the micrometre to the centimetre tumour scale. We review recent examples that incorporate experimental data into model parameters. We show that recent mathematical modelling predicts that transport limitations of cell nutrients, oxygen and growth factors may result in cell death that leads to morphological instability, providing a mechanism for invasion via tumour fingering and fragmentation. These conditions induce selection pressure for cell survivability, and may lead to additional genetic mutations. Mathematical modelling further shows that parameters that control the tumour mass shape also control its ability to invade. Thus, tumour morphology may serve as a predictor of invasiveness and treatment prognosis.
Article
Full-text available
In this article, we present a new multiscale mathematical model for solid tumour growth which couples an improved model of tumour invasion with a model of tumour-induced angiogenesis. We perform nonlinear simulations of the ulti-scale model that demonstrate the importance of the coupling between the development and remodeling of the vascular network, the blood flow through the network and the tumour progression. Consistent with clinical observations, the hydrostatic stress generated by tumour cell proliferation shuts down large portions of the vascular network dramatically affecting the flow, the subsequent network remodeling, the delivery of nutrients to the tumour and the subsequent tumour progression. In addition, extracellular matrix degradation by tumour cells is seen to have a dramatic affect on both the development of the vascular network and the growth response of the tumour. In particular, the newly developing vessels tend to encapsulate, rather than penetrate, the tumour and are thus less effective in delivering nutrients. KeywordsSolid tumour–Avascular growth–Angiogenesis–Vascular growth–Multiscale mathematical model
Article
Full-text available
Tissue engineered bone grafts are among the most promising approaches to heal large bone defects. An in vitro culture phase prior to transplantation provides the opportunity to optimize the graft properties. In our study we developed computational models for the investigation of the deformation, perfusion and revascularization of a porous β-tricalcium phosphate (β-TCP) scaffold seeded with human bone marrow derived mesenchymal stem cells (MSCs). The deformation model allows predicting the resulting forces in the β-TCP scaffold due to different external loadings. In addition to pressure related forces, fluid convection within the scaffold also exerts shear stress onto the cells. However, perfusion is necessary for the supply of the cells with nutrients such as glucose. To find an optimal ratio between shear stress and nutrients supply, we develop fluidic models for the β-TCP scaffold. Furthermore to the scaffold specifications, also the cellular glucose consumption of osteogenic differentiated and undifferentiated MSCs was integrated in the computational model. Beyond the in vitro culture phase, bone grafts have to be supplied by the host ’ s vascular system. Therefore angiogenesis has to be induced, e.g. by preloading the graft with pro angiogenic factors such as VEGF-A. A stochastic model, based on the Fokker-Planck equation was developed to investigate the impact of a given cytokine gradient onto the endothelial cell migration. The stochastic model was parameterized by data derived from live cell imaging studies.
Article
Significant progress has been made over the years in the development of in vitro-engineered substitutes that mimic human skin, either to be used as grafts for the replacement of lost skin or for the establishment of human-based in vitro skin models. This review summarizes these advances in in vivo and in vitro applications of tissue-engineered skin. We further highlight novel efforts in the design of complex disease-in-a-dish models for studies ranging from disease etiology to drug development and screening.
Article
Full-text available
Carcinogenesis is a highly complex, multi-stage process that can occur over a relatively long period before its clinical manifestation. While the sequence in which a cancer cell acquires the necessary traits for tumour formation can vary, there are a number of mechanisms that are common to most, if not all, cancers across the spectrum of possible causes. Many aspects of carcinogenesis can be modelled in vitro. This has led to the development of a number of mechanistically driven, cell-based assays to assess the pro-carcinogenic and anti-carcinogenic potential of chemicals. A review is presented of the current in vitro models that can be used to study carcinogenesis, with examples of cigarette smoke testing in some of these models, in order to illustrate their potential applications. We present an overview of the assays used in regulatory genotoxicity testing, as well as those designed to model other aspects that are considered to be hallmarks of cancer. The latter assays are described with a view to demonstrating the recent advances in these areas, to a point where they should now be considered for inclusion in an overall testing strategy for chemical carcinogens.
Article
Significant progress has been made over the years in the development of in vitro-engineered substitutes that mimic human skin, either to be used as grafts for the replacement of lost skin or for the establishment of human-based in vitro skin models. This review summarizes these advances in in vivo and in vitro applications of tissue-engineered skin. We further highlight novel efforts in the design of complex disease-in-a-dish models for studies ranging from disease etiology to drug development and screening.
Article
Hepatocyte growth factor (HGF) and its receptor play an important role in the formation and progression of glioma and can promote tumor proliferation. In this study, we investigated the ability of HGF to promote the proliferation and invasion of U251n cells; we also tested the effects of HGF on stromal cell-derived factor 1 (SDF1) and CXCR4 mRNA expression. We measured the effect of HGF on the proliferation of U251n cells using enzyme-linked immunosorbent assays (ELISAs) to detect incorporated bromodeoxyuridine (BrdU) as a marker of DNA synthesis. The effects of HGF and SDF-1 on U251n cell invasion and proliferation were measured using the inhibitors K252a to c-Met and AMD3100 to CXCR4. SDF-1 and CXCR4 mRNA and protein expression were measured using quantitative polymerase chain reaction (PCR) and fluorescence-activated cell sorter (FACS) analysis. Small interfering (si)RNAs were also used to down-regulate HGF and c-Met expression in U251n cells. HGF significantly increased U251n cell proliferation and invasion in a dose-dependent manner; K252a blocked this. AMD3100 blocked invasion but not proliferation. CXCR4 and SDF-1 mRNAs were up-regulated when cells were treated with HGF. CXCR4 and SDF-1 mRNA levels and HGF and c-Met protein levels were down-regulated after cells were transfected with siRNAs. HGF has a direct effect on glioma cell proliferation and invasion. HGF up-regulates SDF-1 and CXCR4 mRNA expression and contributes to cell invasion.
Article
Cardiovascular tissue engineering is a fast evolving field of biomedical science and technology to manufacture viable blood vessels, heart valves, myocar-dial substitutes and vascularised complex tissues. In consideration of the specific role of the haemodynamics of human circulation, bioreactors are a fundamental of this field. The development of perfusion bioreactor technology is a consequence of successes in extracorporeal circulation techniques, to provide an in vitro environment mimicking in vivo conditions. The bioreactor system should enable an automatic hydrodynamic regime control. Furthermore, the systematic studies regarding the cellular responses to various mechanical and biochemical cues guarantee the viability, bio-monitoring, testing, storage and transportation of the growing tissue. The basic principles of a bioreactor used for cardiovascular tissue engineering are summarised in this chapter.
Article
Full-text available
We report a porous membrane-based cell culture device that can conduct localized electrical stimulation of a cell monolayer. The device's cell culture substrate is a microporous alumina membrane with an underlying thin poly(dimethylsiloxane) (PDMS) film spotted with holes. When electric current is generated between the device's Pt ring electrodes--one of which is placed above the cells and the other below the PDMS layer--the current density condenses at the holes in the PDMS film, and cells located above the holes can be electrically stimulated. C2C12 cells were confluently cultured on the substrate and were differentiated to myotubes. To control the stimulated area in the substrate, we attempted to seal and reopen the holes of the PDMS film by using an air bubble. Since the current pulse could be effectively blocked at the sealed holes, fluorescent Ca2+ transients, indicative of cellular excitation, were observed from the myotubes located above holes in the open state.
Article
Liver tissue that is functional and viable for several weeks in vitro represents an auspicious test system for basic and applied research. In this study, a coculture system for hepatocytes (HCs) and microvascular endothelial cells (mECs) was generated applying tissue-engineering techniques, establishing the basis for a new bioartificial liver in vitro model. Porcine mECs were seeded on a decellularized porcine jejunal segment with preserved vascular structures. Porcine HCs were seeded onto this vascularized scaffold, and the resulting coculture was maintained for 3 weeks in vitro. Tissue morphology and differentiation was monitored using histology and immunohistochemistry. Tissue metabolism was monitored using daily assessment of urea and lactate production. HC monolayer cultures served as controls. The 2-stage seeding procedure resulted in a 3-dimensional coculture system harboring HC cell clusters in multiple cell layers lining the generated mEC-seeded capillary structures. It was viable for 3 weeks, and HCs maintained their morphology and differentiation. Biochemical testing revealed stable metabolic activity of the tissue culture. In contrast, HCs cultured in monolayer showed morphological dedifferentiation and an unfavorable metabolic state. Our mEC-HC coculture represents a new approach toward a functional bioartificial liver-like tissue applicable as a test system for basic and applied research.
Article
Full-text available
The growth of tumour cells as three-dimensional multicellular spheroids in vitro has led to important insights in tumour biology, since properties of the in vivo-tumour such as proliferation or nutrient gradients, can be studied under controlled conditions. While this review starts with an update of recent data on spheroid monocultures, especially concerning tumour microenvironment and therapeutic modalities, the main emphasis is put on the spectrum of heterologous cultures which have evolved in previous years. This type of culture includes tumour cell interaction with endothelial, fibroblast or immunocompetent cells. The relation of the spheroid culture model to other types of three-dimensional culture and our critical evaluation and presentation of the technical aspects of growing and analysing spheroids are included in the text. These topics are chosed to help the experimental pathologist design experiments with tumour spheroids and to stimulate discussion.
Article
Full-text available
Optimized in vitro formation of strong tissue is a prerequisite for tissue engineering of cardiovascular structures, such as heart valves and blood vessels. This study evaluates different growth media additives as to cell proliferation, extracellular matrix formation, and mechanical characteristics. Biodegradable polymers were seeded with human vascular myofibroblasts. Group A was cultured with standard medium, groups B, C, and D were in addition supplemented with ascorbate, fibroblast growth factor (bFGF), and both respectively. Analysis included histology, electron microsocopy, mechanical testing, and biochemical assays for cell proliferation (DNA) and extracellular matrix (collagen). DNA content increased in all groups, showing significantly more cells in group C and D after 14d. Collagen increased in all groups, except for C. Morphology showed viable, layered cellular tissue, with collagen fibrils after 2w, most pronounced in B and D. Mechanical properties decreased initially, stabilizing after 2w. In conclusion, standard nutrient media were efficient for seeded human vascular cells cultured on biodegradable meshes. Supplementation with bFGF+ascorbate resulted in enhanced early cell proliferation and structurally more mature tissue formation.
Article
Full-text available
The individual and synergistic contributions of two transcription factors, EFG1 and CPH1, have been characterized with regard to adhesion to, and invasion of, human epithelia by Candida albicans. For this purpose two in vitro reconstructed tissue models were developed. A multi-layered model of human epidermis was used to simulate superficial infections of the skin, whereas a reconstructed human intestinal model was used to mimic the first steps of systemic infections. It was shown that C. albicans deleted for both transcription factors CPH1 and EFG1, in contrast to the congenic clinical isolate Sc5314, was neither able to adhere to, nor to penetrate, either of the model systems. A strain deleted for EFG1 alone showed significant reduction in adhesion and was not able to penetrate through the stratum corneum. However, strains deleted for CPH1 showed phenotypes paralleling the phenotypes of the clinical isolate Sc5314. Using different types of multi-layered human tissues reconstructed in vitro the individual contributions of Efg1p and Cph1p to two important virulence factors of C. albicans, namely adhesion and invasion, could be defined.
Article
Full-text available
We report here that cells co-purifying with mesenchymal stem cells--termed here multipotent adult progenitor cells or MAPCs--differentiate, at the single cell level, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. When injected into an early blastocyst, single MAPCs contribute to most, if not all, somatic cell types. On transplantation into a non-irradiated host, MAPCs engraft and differentiate to the haematopoietic lineage, in addition to the epithelium of liver, lung and gut. Engraftment in the haematopoietic system as well as the gastrointestinal tract is increased when MAPCs are transplanted in a minimally irradiated host. As MAPCs proliferate extensively without obvious senescence or loss of differentiation potential, they may be an ideal cell source for therapy of inherited or degenerative diseases.
Article
Full-text available
Angiogenesis, the process that leads to the formation of new blood vessels or neovascularization, continues to be a topic of major scientific and public interest. As knowledge of the molecular mechanisms that regulate neovascularization continues to emerge, there is increasing hope that new discoveries will lead to newer therapies that target angiogenesis as a reliable option for disease therapy. For example, it may be possible to develop strategies that, on the one hand, are designed to limit angiogenesis for the treatment of chronic diseases such as cancer or rheumatoid arthritis and, on the other, to promote angiogenesis in the ischemic heart or diabetic limb. With the emergence of tissue engineering as a discipline, it has become increasingly clear that long-term success in organ and tissue reconstruction will depend on the ability to develop a stable, renewable supply of blood vessels. In this review, I will provide a brief overview of this remarkably versatile biological response and discuss how recent discoveries in the field of angiogenesis have influenced the development of novel therapies, forced a reconsideration of conventional therapies, and revolutionized approaches to organ and tissue reconstruction.
Article
Full-text available
Biological scaffolds exhibit advantageous properties for tissue engineering of small diameter vessels. The influence of their extracellular matrix (ECM) components during in vivo repopulation is unknown. We implanted different xenogenic vascular matrices in a rat model to determine the influence of scaffold-thickness and ECM composition on in vivo repopulation. Decellularized ovine jugular vein (JV, n=42), carotid artery (CA, n=42) and aorta (AO, n=42) were implanted subcutaneously in the neck of adult male rats. Animals were sacrificed 2, 4 and 8 weeks after implantation. Cell and matrix morphology of explanted scaffolds were characterized by hematoxylin-eosin and pentachrome staining. Monoclonal anti-rat-CD31 was used to identify revascularization. Quantification of cell density was done by DNA-isolation. THICKNESS OF IMPLANTED XENOGENIC SCAFFOLDS VARIED ACCORDING TO THE MATERIAL USED (AO: 3.0-3.8mm; CA: 0.7-0.88mm; JV: 0.35-0.61mm). Immunohistology revealed complete repopulation of AO, CA, and JV scaffolds with endothelial cells and myofibroblasts within 2 weeks. After 8 weeks of implantation, AO scaffolds were completely covered by an endothelial monolayer and showed signs of a central matrix degeneration. JV scaffolds were completely degenerated at this stage. In contrast, CA scaffolds showed preserved ECM with a normal myofibroblast population and endothelial cell coverage.
Article
Full-text available
Targeted gene disruption in mice is a powerful tool for generating murine models for human development and disease. While the human genome program has helped to generate numerous candidate genes, few genes have been characterized for their precise in vivo functions. Gene targeting has had an enormous impact on our ability to delineate the functional roles of these genes. Many gene knockout mouse models faithfully mimic the phenotypes of the human diseases. Because some models display an unexpected or no phenotype, controversy has arisen about the value of gene-targeting strategies. We argue in favor of gene-targeting strategies, provided they are used with caution, particularly in interpreting phenotypes in craniofacial and oral biology, where many genes have pleiotropic roles. The potential pitfalls are outweighed by the unique opportunities for developing and testing different therapeutic strategies before they are introduced into the clinic. In the future, we believe that genetically engineered animal models will be indispensable for gaining important insights into the molecular mechanisms underlying development, as well as disease pathogenesis, diagnosis, prevention, and treatment.
Article
Full-text available
Compounds can be screened for pharmaceutical activity either by detecting interactions with specified target molecules such as receptors or enzymes (molecular screening) or observing effects on the structure or physiological activities of cells or tissues (phenotypic screening). Screening at the molecular level has been greatly enhanced by fluorescence methods. Especially the combination of confocal detection with measurements of the amplitudes and time courses of fluorescence fluctuations have reduced sample volumes to < microliters and have increased throughputs to >100000 compounds per day. Screening at the molecular level, however, does not provide information about the effects of test compounds on cellular functions. Phenotypic screening, although much slower than molecular screening, does provide information about effects on cell or tissue structure or function and therefore can be used to eliminate at an early stage compounds that are toxic or do not produce the desired cellular response. Tissue constructs reconstituted using cells of specified types and defined extracellular matrix components provide test systems for detecting the effects of test compounds on cellular mechanical functions such as the development of contractile force and on cell and matrix structure and stiffness. For example, constructs based on vascular smooth muscle cells provide information about effects on cellular contractile force that can be used to identify agents that control blood pressure. Tissue constructs that mimic skeletal, smooth and heart muscles and connective tissues have been produced and can be used to study mechanical and structural responses to active compounds.
Article
Full-text available
Over the past few years, establishment and adaptation of cell-based assays for drug development and testing has become an important topic in high-throughput screening (HTS). Most new assays are designed to rapidly detect specific cellular effects reflecting action at various targets. However, although more complex than cell-free biochemical test systems, HTS assays using monolayer or suspension cultures still reflect a highly artificial cellular environment and may thus have limited predictive value for the clinical efficacy of a compound. Today's strategies for drug discovery and development, be they hypothesis free or mechanism based, require facile, HTS-amenable test systems that mimic the human tissue environment with increasing accuracy in order to optimize preclinical and preanimal selection of the most active molecules from a large pool of potential effectors, for example, against solid tumors. Indeed, it is recognized that 3-dimensional cell culture systems better reflect the in vivo behavior of most cell types. However, these 3-D test systems have not yet been incorporated into mainstream drug development operations. This article addresses the relevance and potential of 3-D in vitro systems for drug development, with a focus on screening for novel antitumor drugs. Examples of 3-D cell models used in cancer research are given, and the advantages and limitations of these systems of intermediate complexity are discussed in comparison with both 2-D culture and in vivo models. The most commonly used 3-D cell culture systems, multicellular spheroids, are emphasized due to their advantages and potential for rapid development as HTS systems. Thus, multicellular tumor spheroids are an ideal basis for the next step in creating HTS assays, which are predictive of in vivo antitumor efficacy.
Article
Full-text available
The cooperation between epithelial and mesenchymal cells is essential for embryonic development and probably plays an important role in pathological phenomena such as wound healing and tumor progression. It is well known that many epithelial tumors are characterized by the local accumulation of connective tissue cells and extracellular material; this phenomenon has been called the stroma reaction. One of the cellular components of the stroma reaction is the myofibroblast, a modulated fibroblast which has acquired the capacity to neoexpress alpha-smooth muscle actin, the actin isoform typical of vascular smooth muscle cells, and to synthesize important amounts of collagen and other extracellular matrix components. It is now well accepted that the myofibroblast is a key cell for the connective tissue remodeling which takes place during wound healing and fibrosis development. Myofibroblasts are capable of remodeling connective tissue but also interact with epithelial cells and other connective tissue cells and may thus control such phenomena as tumor invasion and angiogenesis. In this review we discuss the mechanisms of myofibroblast evolution during fibrotic and malignant conditions and the interaction of myofibroblasts with other cells in order to control tumor progression. On this basis we suggest that the myofibroblast may represent a new important target of antitumor therapy.
Article
Full-text available
We generated a vascularized, autologous, reseeded bladder substitute and evaluated immediate vascularization and perfusion of the graft after implantation to the recipient organism in a porcine model. Acellular matrix was processed from porcine small bowel segments by subsequent mechanical, chemical and enzymatic decellularization, preserving the jejunal arteriovenous pedicles. In 2 separate steps the matrix was reseeded with primary bladder smooth muscle cells (SMCs) and urothelial cells (UCs), and its vascular structures were resurfaced with endothelial progenitor cells (EPCs). To evaluate graft perfusion short-term implantation was performed. The acellular scaffold was successfully repopulated with multilayers of ingrowing SMCs and superficial UCs. After reseeding the jejunal arteriovenous pedicles with EPCs and cultivation for 3 weeks the larger vessels as well as the intramural scaffold capillary network were repopulated with cell monolayers expressing endothelial specific proteins. Perfusion stagnation and implant thrombosis occurred within 30 minutes after the implantation of acellular scaffolds not reseeded with EPCs. In the EPC reseeded group the vascular system revealed intact perfusion and no relevant thrombus formation was observed after 1 or 3 hours. The current study of successful SMC and UC reseeding, vessel resurfacing with EPCs and short-term vascular patency represents the promising in vitro and in vivo basis for further evaluation of this biological vascularized matrix in chronic long-term large animal implantation experiments.
Article
Full-text available
Intestinal myofibroblasts are alpha-smooth muscle actin-positive stromal cells that exist as a syncytium with fibroblasts and mural cells in the lamina propria of the gut. Through expression and secretion of cytokines, chemokines, growth factors, prostaglandins, and basal lamina/extracellular matrix molecules, as well as expression of adhesion molecules and receptors for many of the same soluble factors and matrix, myofibroblasts mediate information flow between the epithelium and the mesenchymal elements of the lamina propria. With the use of these factors and receptors, they play a fundamental role in intestinal organogenesis and in the repair of wounding or disease. Intestinal neoplasms enlist and conscript myofibroblast factors and matrix molecules to promote neoplastic growth, carcinoma invasion, and distant metastases.
Article
Full-text available
Human mesenchymal stem cells (MSCs) are increasingly being considered in cell-based therapeutic strategies for regeneration of various organs/tissues. However, the signals required for their homing and recruitment to injured sites are not yet fully understood. Because stromal-derived factor (SDF)-1 and hepatocyte growth factor (HGF) become up-regulated during tissue/organ damage, in this study we examined whether these factors chemoattract ex vivo-expanded MSCs derived from bone marrow (BM) and umbilical cord blood (CB). Specifically, we investigated the expression by MSCs of CXCR4 and c-met, the cognate receptors of SDF-1 and HGF, and their functionality after early and late passages of MSCs. We also determined whether MSCs express matrix metalloproteinases (MMPs), including membrane type 1 (MT1)-MMP, matrix-degrading enzymes that facilitate the trafficking of hematopoietic stem cells. We maintained expanded BM- or CB-derived MSCs for up to 15-18 passages with monitoring of the expression of 1) various tissue markers (cardiac and skeletal muscle, neural, liver, and endothelial cells), 2) functional CXCR4 and c-met, and 3) MMPs. We found that for up to 15-18 passages, both BM- and CB-derived MSCs 1) express mRNA for cardiac, muscle, neural, and liver markers, as well as the vascular endothelial (VE) marker VE-cadherin; 2) express CXCR4 and c-met receptors and are strongly attracted by SDF-1 and HGF gradients; 3) express MMP-2 and MT1-MMP transcripts and proteins; and 4) are chemo-invasive across the reconstituted basement membrane Matrigel. These in vitro results suggest that the SDF-1-CXCR4 and HGF-c-met axes, along with MMPs, may be involved in recruitment of expanded MSCs to damaged tissues.
Article
Full-text available
Much of the knowledge regarding the regulatory pathways for adult stem cell self-renewal and differentiation has been obtained from the results of in vitro cultures. However, it is unclear if adult stem cells are controlled in the same way under physiological conditions. We examined this issue with respect to the migration of stem cells to tissue injury and how switch from a migratory state to one of proliferation wherein they participate in development. Building on our previous identification of multipotent stem cells in trabecular bone, we have examined the in vitro behavior of these cells within the bone milieu. We found that cell proliferation is inhibited within the trabecular bone niche as cells migrate out of the trabecular bone prior to proliferation. Additionally, multiple cell types were detected in adult trabecular bone, including osteoblasts, osteoclasts, endothelial cells, and Stro-1-positive mesenchymal stem cells. Furthermore, we demonstrated that Stro-1-positive cells migrated out of their native bone niche to generate multipotential stem and progenitor cells during in vitro culture. We conclude that self-renewal and differentiation of adult stem cells in connective tissues are tightly controlled and separately orchestrated processes. A regulatory network of extrinsic factors and intrinsic signals acts to stimulate the exit of stem cells from their niche so that they can localize to sites of wound healing, where they participate in development after functional differentiation.
Article
The overall success rate nationally in treating esophageal carcinomas remains poor, with over 90% of patients succumbing to the disease. In part I of this two-part series, we explored epidemiology, presentation and progression, work-up, and surgical approaches. In part II, we explore the promising suggestions of integrating chemotherapy and radiation therapy into the multimodal management of esophageal cancers. Alternative approaches to resection alone have been sought because of the overall poor survival rates of esophageal cancer patients, with failures occurring both local-regionally and distantly. Concomitant chemotherapy and radiation therapy (XRT) have been shown, by randomized trial, to be more effective than XRT alone in treating unresectable esophageal cancers and also have shown promise as a neoadjuvant treatment when combined with surgery in the multimodal treatment of this disease. Various studies have also addressed issues such as preoperative chemotherapy, radiation dose escalation, chemotherapy/XRT as a definitive treatment versus use as a surgical adjuvant, and alternative chemotherapy regimens. There are suggestions of some progress, but this remains a difficult problem area in which management is continuing to evolve.
Article
Zusammenfassung Es wird über die Xenotransplantation von 216 malignen Ovarialtumoren auf thymusaplastische nu/nu-Mäuse (NMRI) berichtet. Nach histologischen und metrischen Gesichtspunkten sind 76&percnt; der Trans-plantate angewachsen. Von diesen sind weitere Passagen in 40&percnt; möglich, serienmäßig transplantierbare Zellinien in 20&percnt;. Der humane Ursprung der Xenotransplantate ist durch Isoenzymuntersuchungen belegt, allerdings zeigen sich Änderungen des Proliferationsverhaltens und einzelner Tumormerkmale im Verlaufe der Passagen gegenüber dem ursprünglichen Patientengewebe. Mit der grundsätzlichen Möglichkeit, menschliches Tumorgewebe unbegrenzt in vivo zur Verfügung zu haben, nimmt die nu/nu-Maus eine Sonderstellung ein und stellt ein wichtiges Bindeglied bei der präklinischen Testung zwischen Tiertumoren und Patienten dar. Für eine Individualtestung im Sinne eines ‘predictive assay’ ist das Modell aufgrund des nicht vorhersehbaren, mangelhaften Wachstums weniger geeignet.Copyright © 1984 S. Karger AG, Basel
Article
The loss or failure of an organ or tissue is one of the most frequent, devastating, and costly problems in human health care. A new field, tissue engineering, applies the principles of biology and engineering to the development of functional substitutes for damaged tissue. This article discusses the foundations and challenges of this interdisciplinary field and its attempts to provide solutions to tissue creation and repair.
Article
This article reviews actual advances in the development and application of three-dimensional (3-D) cell culture systems. Recent therapeutically oriented studies include characterization of multicellular-mediated drug resistance, novel ways of quantifying hypoxia, and new approaches to more efficient immunotherapy. Recent progress toward understanding the development of necrosis in tumor spheroids has been made using novel spheroid models. 3-D cultures have been used for studies on molecular mechanisms involved in invasion and metastasis, with a major focus on the role of E-cadherin. Similarly, tumor angiogenesis and the significance of vascular endothelial growth factor have been investigated in a variety of 3-D culture systems. There are many ongoing developments in tissue modeling or remodeling that promise significant progress toward the development of bioartificial liver support and artificial blood. Perhaps one of the most interesting areas of basic research with 3-D cultures is the characterization of embryoid bodies obtained from stable embryonic stem cells. These models have greatly increased the understanding of embryonic development, in particular through the notable exceptional advances in cardiogenesis.
Article
In vitro liver preparations are increasingly used for the study of hepatotoxicity of chemicals. In recent years their actual advantages and limitations have been better defined. The cell models, slices, and mainly primary hepatocyte cultures, appear to be the most powerful in vitro systems, as liver-specific functions and responsiveness to inducers are retained either for a few days or several weeks depending on culture conditions. Maintenance of phase I and phase II xenobiotic metabolizing enzyme activities allows various chemical investigations to be performed, including determination of kinetic parameters, metabolic profile, interspecies comparison, inhibition and induction effects, and drug-drug interactions. In vitro liver cell models also have various applications in toxicology: screening of cytotoxic and genotoxic compounds, evaluation of chemoprotective agents, and determination of characteristic liver lesions and associated biochemical mechanisms induced by toxic compounds. Extrapolation of the results to the in vivo situation remains a matter of debate. Presently, the most convincing applications of liver cell models are the studies on different aspects of metabolism and mechanisms of toxicity. For the future, there is a need for better culture conditions and differentiated hepatocyte cell lines to overcome the limited availability of human liver tissues. In addition, strategies for in vitro analysis of potentially toxic chemicals must be better defined.
Article
The trend towards assay miniaturization for high-throughput and ultra-high-throughput screening continues to spur development of homogeneous, fluorescence-based assays in higher density, smaller volume microplate formats. Recently, first-generation microfluidic devices have been designed for performing continuous-flow biochemical and cell-based assays. These devices provide orders-of-magnitude reduction in reagent consumption, and offer the potential for implementing high-throughput screening in formats that integrate up-front compound handling with unique assay functionality.
Article
Recent research using multicellular tumor spheroids has resulted in new insights in the regulation of invasion and metastasis, angiogenesis and cell cycle kinetics. The onset and expansion of central necrosis in tumor spheroids has been characterized to be a complex interaction of several mechanisms; in a number of cases, necrosis is not a consequence of hypoxia or anoxia, but emerges as secondary necrosis following an accumulation of apoptosis in spheroids. Recent therapeutically oriented studies have been directed towards novel hypoxic markers, targeted therapy, multicellular-mediated drug resistance, and heavy ion irradiation of spheroids. Research efforts should be enhanced mainly in the fields of tumor tissue modeling by heterotypic three-dimensional (3D) cultures and of apoptotic versus necrotic cell death. Based on the fundamental differences between monolayer and 3D cultures, spheroids should become mandatory test systems in therapeutic screening programs.
Article
It has recently been established that the microenvironment plays a major role in many physiological and pathological events. Indeed cell-cell and cell-extracellular matrix contacts are necessary for much cellular function such as differentiation, proliferation, cell death, apoptosis and angiogenesis. For growth, proliferating tumour cells need to be fed by nutrients and oxygen brought by new vessels. In this context, scientists seek a new model that allows for the investigation of both angiogenesis and the influence of the microenvironment on this phenomenon. The purpose of this paper is to review the literature on the relation between tumour and endothelial cells grown as spheroids, a technique that allows us to study in three-dimensions the influence of cell contact on this growth. For the purpose of clarification, this review has recategorised the different studies on spheroids into three classes: (1) spheroids grown in vitro and then reimplanted in animals to follow endothelial cell infiltration; (2) spheroids grown in vitro and then cultured on endothelial cell monolayers; (3) tumours grown in vitro such as organotypic culture. This review attempts to demonstrate that spheroid cell cultures are useful for studying the relation between tumour and endothelial cells and to analyse physiological phenomena such as wound healing, extravasation and intravasation.
Article
An analysis of the activity of compounds tested in pre-clinical in vivo and in vitro assays by the National Cancer Institute's Developmental Therapeutics Program was performed. For 39 agents with both xenograft data and Phase II clinical trials results available, in vivo activity in a particular histology in a tumour model did not closely correlate with activity in the same human cancer histology, casting doubt on the correspondence of the pre-clinical models to clinical results. However, for compounds with in vivo activity in at least one-third of tested xenograft models, there was correlation with ultimate activity in at least some Phase II trials. Thus, an efficient means of predicting activity in vivo models remains desirable for compounds with anti-proliferative activity in vitro. For 564 compounds tested in the hollow fibre assay which were also tested against in vivo tumour models, the likelihood of finding xenograft activity in at least one-third of the in vivo models tested rose with increasing intraperitoneal hollow fibre activity, from 8% for all compounds tested to 20% in agents with evidence of response in more than 6 intraperitoneal fibres (P< 0.0001). Intraperitoneal hollow fibre activity was also found to be a better predictor of xenograft activity than either subcutaneous hollow fibre activity or intraperitoneal plus subcutaneous activity combined. Since hollow fibre activity was a useful indicator of potential in vivo response, correlates with hollow fibre activity were examined for 2304 compounds tested in both the NCI 60 cell line in vitro cancer drug screen and hollow fibre assay. A positive correlation was found for histologic selectivity between in vitro and hollow fibre responses. The most striking correlation was between potency in the 60 cell line screen and hollow fibre activity; 56% of compounds with mean 50% growth inhibition below 10(-7.5) M were active in more than 6 intraperitoneal fibres whereas only 4% of compounds with a potency of 10(-4) M achieved the same level of hollow fibre activity (P< 0.0001). Structural parameters of the drugs analysed included compound molecular weight and hydrogen-bonding factors, both of which were found to be predictive of hollow fibre activity.
Article
Tissue engineering has emerged as a rapidly expanding approach to address the organ shortage problem. It is an "interdisciplinary field that applies the principles and methods of engineering and the life sciences toward the development of biological substitutes that can restore, maintain, or improve tissue function." Much progress has been made in the tissue engineering of structures relevant to cardiothoracic surgery, including heart valves, blood vessels, myocardium, esophagus, and trachea. (C) 2001 by The Society of Thoracic Surgeons.
Article
The completion of the Human Genome Project has signaled the beginning of the post-genome era, with a corresponding shift in focus from the sequencing and identification of genes to the exploration of gene function. A rate-limiting step in deriving value from this gene sequence information is determining the potential pharmaceutical applications of genes and their encoded proteins. This validation step is crucial for focusing efforts and resources on only the most promising targets. Strategies using reverse mouse genetics provide excellent methods for validating potential targets and therapeutic proteins in vivo in a mammalian model system.
Article
Recent progress in the biology of cell adhesion is enabling cell culture models to better reproduce in vivo functions. Cues from adhesion to extracellular matrix and neighboring cells are important regulators of cell behaviors. The recent adaptation of semiconductor tools to spatially organize cells and their adhesions has enhanced our ability to engineer cell functions ex vivo. By using these tools to create more in vivo-like cultures, cell-based drug discovery and target validation could be improved. This review explores the biological advances made by these microfabrication tools and discusses how they could enable high-throughput cell-based assays.
Article
The opportunity provided by the Albert Lasker Award for Special Achievement in Medical Science to reflect on my 'career' does not, I am sure, give me license to dwell on the humid, sweltering summers of a childhood in Mississippi. But perhaps I may be permitted to say a brief word of thanks to my teachers at the University of Mississippi, especially Dean Parker, a Drosophila geneticist of the T.S. Painter School, and Robert Glaser, at the time an assistant professor of medicine at Washington University School of Medicine. Parker taught us what a gene was and how to observe its effects, but it was in Robert Glaser's rheumatic fever laboratory that I tried to do my first experiments. A colleague, Stephen Morse, and I infected rabbits in their tonsils with Group A streptococci in an effort, not completely unsuccessful, to mimic the secondary heart muscle damage that occurs in rheumatic fever as a sequel to infection by Group A streptococci1.
After disappointing results achieved with older chemosensitivity tests such as the human tumor clonogenic assay (HTCA) during the 1980s, the last decade has seen a renaissance of the concept of individualized chemotherapy in oncology, markedly stimulated by the development of newer nonclonogenic assays. These methods appear to be able to overcome major technical limitations associated with older assays, now allowing for successful testing of most of the tumor specimens submitted. Currently, the ATP-based tumor chemosensitivity assay (ATP-TCA) can be regarded as the most sophisticated assay to investigate both solid samples and effusions derived from patients with various organ tumors. During the last 5 years, the ATP-TCA has been used successfully to screen for novel drug combinations for further clinical use in both ovarian and breast cancer such as mitoxantrone plus paclitaxel (NT) and treosulfan plus gemcitabine (TG), respectively. Clinical trials that have been set up in heavily pretreated patients with recurrent ovarian or breast cancer have convincingly confirmed the high activity of these combinations previously demonstrated in preclinical investigations using the ATP-TCA. In a recent phase II trial performed in 59 patients with relapsed ovarian carcinoma, |ATP-TCA-directed therapy was able to triple the response rate and to double the survival time, compared with published empirical chemotherapy regimes. Preliminary results with ATP-TCA-directed therapy in breast cancer also evidenced promising response rates. These results have been confirmed by additional prospective clinical trials using other types of modern nonclonogenic assays. A phase III trial that is now actively recruiting patients with platinum-refractory ovarian cancer to verify the promising phase II studies will prove the further value of the ATP-TCA as a predictor applicable in routine clinical oncology.
Article
In order to translate the findings from basic cellular research into clinical applications, cell-based models need to recapitulate both the 3D organization and multicellular complexity of an organ but at the same time accommodate systematic experimental intervention. Here we describe a hierarchy of tractable 3D models that range in complexity from organotypic 3D cultures (both monotypic and multicellular) to animal-based recombinations in vivo. Implementation of these physiologically relevant models, illustrated here in the context of human epithelial tissues, has enabled the study of intrinsic cell regulation pathways and also has provided compelling evidence for the role of the stromal compartment in directing epithelial cell function and dysfunction. Furthermore the experimental accessibility afforded by these tissue-specific 3D models has implications for the design and development of cancer therapies.
Article
The maturation of nascent vasculature, formed by vasculogenesis or angiogenesis, requires recruitment of mural cells, generation of an extracellular matrix and specialization of the vessel wall for structural support and regulation of vessel function. In addition, the vascular network must be organized so that all the parenchymal cells receive adequate nutrients. All of these processes are orchestrated by physical forces as well as by a constellation of ligands and receptors whose spatio-temporal patterns of expression and concentration are tightly regulated. Inappropriate levels of these physical forces or molecules produce an abnormal vasculature--a hallmark of various pathologies. Normalization of the abnormal vasculature can facilitate drug delivery to tumors and formation of a mature vasculature can help realize the promise of therapeutic angiogenesis and tissue engineering.
Article
There's a big difference between a flat layer of cells and a complex, three-dimensional tissue. But until recently, many biologists have glossed over this fact. Alison Abbott discovers what they've been missing.
Article
Tissue development, homeostasis and tumor pathogenesis all depend upon a complex dialogue between multiple cell types operating within a dynamic three-dimensional (3D) tissue extracellular matrix microenvironment. A major issue is whether the spatial organization of a cell within this 3D tissue microenvironment could modulate cell responsiveness to regulate cell fate decisions such as survival, and if so how. Classic developmental model systems and transgenic animals are instructive but pose special challenges for investigators conducting signaling studies and biochemical assays in tissues. As an alternative, 3D culture model systems exist in which cell-adhesion dependent tissue architecture, heterotypic cell-cell interactions and tissue differentiation can be recapitulated with good fidelity. 3D cell culture models are slowly revealing how tissue architecture can dramatically influence how a cell responds to exogenous stimuli to modify its apoptotic behavior and hence should prove instrumental for identifying novel cell death pathways.
Article
Transplantation of a human trachea has been reported only twice in the literature with limited documentation of the functional and structural properties of the allograft. A 57-year-old patient with chronic obstructive pulmonary disease with low segment tracheal stenosis was accepted for lung transplantation and 2-stage tracheal allotransplantation. Standard bilateral sequential lung transplantation was performed with the transfer of the donor trachea into the recipient's abdomen, which was wrapped in the greater omentum and sutured into the abdominal wall, similar to a stoma. The patient received immunosuppression consisting of cyclosporine A, mycophenolate mofetil, and cortisone. Sixty days later, the tracheal allograft presented with macroscopically normal appearance with maintained elasticity and rigidity. The patient underwent a cricotracheal resection 6 months after lung transplantation. However, reconstruction with direct end-to-end anastomosis was achievable. The tracheal allograft, therefore not needed for reconstruction, was harvested and underwent complete investigations. Cross-section of the graft revealed a mechanically stable and macroscopically intact trachea. Hematoxylin-eosin staining demonstrated vital cartilage covered by respiratory epithelium. Angiography, followed by corrosion studies and electromicroscopy, demonstrated excellent vascularization of the tracheal wall. The patient is alive 31 months posttransplantation and remains in bronchiolitis obliterans syndrome stage 0. Human trachea wrapped in omentum maintains its functional and structural integrity and may be used for 2-stage allotransplantation.
Article
Patients suffering from diseased and injured organs may be treated with transplanted organs. However, there is a severe shortage of donor organs that is worsening yearly given the aging population. Scientists in the field of regenerative medicine and tissue engineering apply the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Therapeutic cloning, where the nucleus from a donor cell is transferred into an enucleated oocyte in order to extract pluripotent embryonic stem cells, offers a potentially limitless source of cells for tissue engineering applications. The stem cell field is also advancing rapidly, opening new options for therapy. This paper reviews recent advances that have occurred in regenerative medicine and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure.
Article
The clinical goals of tissue engineering are to restore, repair, or replace damaged or lost tissues in the body. Significant progress has been made in recent years, which includes the use of cells or polymer scaffolds as well as combinations of cells and polymers for engineering three-dimensional tissue constructs. However, major challenges still need to be addressed in order for these studies to progress into their clinical applications. The challenges include (1) developing functional polymers, (2) exploring more sources of human cells, and (3) finding ways to keep the engineered construct viable in vitro and in vivo. In addition to clinical applications, tissue engineering can provide new tools for studying cell and developmental biology by providing approaches for cell and tissue growth in three-dimensional environments. In this review we describe recent attempts in addressing some of the challenges of tissue engineering and discuss how such approaches may provide new insights into regulation of cell growth and differentiation.
Article
Oesophageal cancer, in particular adenocarcinomas, has shown a rapid and largely unexplained increase in incidence in the Western world. Despite advances in diagnostic and surgical techniques and improved pre- and postoperative care, the prognosis of most patients is poor. This Review will focus on the use of chemotherapy as part of multimodal treatment and for patients with metastatic disease. Randomised phase III trials have, for the most part, failed to demonstrate a survival advantage with the use of chemotherapy. It must be emphasised that many of these phase III trial were underpowered and do not meet today's standards. Recent phase II trials have suggested some progress when chemotherapy is incorporated into the management of patients with oesophageal cancer. However, confirmatory and adequately powered and designed phase III studies are urgently needed to improve patient outcomes and for better palliation of symptoms.
Article
After myocardial infarction, injured cardiomyocytes are replaced by fibrotic tissue promoting the development of heart failure. Cell transplantation has emerged as a potential therapy and stem cells may be an important and powerful cellular source. Embryonic stem cells can differentiate into true cardiomyocytes, making them in principle an unlimited source of transplantable cells for cardiac repair, although immunological and ethical constraints exist. Somatic stem cells are an attractive option to explore for transplantation as they are autologous, but their differentiation potential is more restricted than embryonic stem cells. Currently, the major sources of somatic cells used for basic research and in clinical trials originate from the bone marrow. The differentiation capacity of different populations of bone marrow-derived stem cells into cardiomyocytes has been studied intensively. The results are rather confusing and difficult to compare, since different isolation and identification methods have been used to determine the cell population studied. To date, only mesenchymal stem cells seem to form cardiomyocytes, and only a small percentage of this population will do so in vitro or in vivo. A newly identified cell population isolated from cardiac tissue, called cardiac progenitor cells, holds great potential for cardiac regeneration. Here we discuss the potential of the different cell populations and their usefulness in stem cell based therapy to repair the damaged heart.
Article
The central nervous system (CNS) and the heart muscle regenerate poorly after injury, yet evidence is mounting that both harbor cells capable of rebuilding neural and cardiac tissue. The reason for the poor regenerative response of CNS tissue and myocardium must therefore lie in the nature of the injury environment, which promotes fibrosis over regeneration. Strategies for regenerating these tissues thus rely on overcoming the fibrotic response by filling lesions with tissue-specific regeneration-competent cells that replace or rescue dying cells, or by activating endogenous regeneration-competent cells that do likewise. There has also been considerable excitement about the possibility of transplanting bone marrow cells into CNS or cardiac lesions to repair them, because bone marrow cells have been reported to be pluripotent. In this chapter, contemporary evidence for the existence of regeneration-competent cells in the CNS and heart is discussed, as well as attempts to use these cells and bone marrow cells to reconstitute new tissue.
Article
Patients suffering from a variety of urologic diseases may be treated with transplanted tissues and organs. However, there is a shortage of donor tissues and organs, which is worsening yearly owing to the ageing population. Scientists in the field of regenerative medicine and tissue engineering are applying the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured urologic tissues. This chapter reviews recent advances that have occurred in the regeneration of urologic organs and describes how these applications may offer novel therapies for patients with urologic disease.
Article
Tissue engineering is an emerging field in regenerative medicine to overcome the problem of end-stage organ failure. However, complex tissues and organs need a vascular supply to guaranty graft survival and render bioartificial organ function. Here we developed methods to decellularize porcine small bowl segments and repopulate the remaining venous and arterial tubular structures within these matrices with allogeneic porcine endothelial progenitor cells. Cellular adherence and vitality was characterized by quantitative 2-[18F]-fluoro-2'-desoxy-glucose (FDG) positron emission tomography (PET) and subsequent immunohistological work up. The generated matrices showed insulin-dependent FDG uptake predominantly in the region of the former vascular structures. Stain for vitality and the specific endothelial markers CD31, VE-Cadherin and Flk-1 matched this functional finding. Providing evidence for vitality up to 3 weeks post reconstitution and typical endothelial differentiation, these results indicate that our generated matrix allows the generation of complex bioartificial tissues and organs for experimental and future clinical application.
Article
Preclinical studies have suggested antitumor activity of an epidermal growth factor (EGF)-receptor targeted therapy with selective tyrosine kinase inhibitors alone or in combination with conventional cytostatic drugs. However, in non-small cell lung cancer (NSCLC), addition of ZD1839 (Iressa) to combination chemotherapy did not improve the therapeutic outcome. Thus, further work is necessary to define factors predicting outcome of combination therapy. In the present study, the activity of ZD1839 alone or in combination with oxaliplatin (Eloxatin) was evaluated in 12 human cancer cell lines including colon, testicular, anaplastic thyroid and epidermoid carcinoma cells. The EGF-receptor protein was overexpressed in line A431 (epidermoid carcinoma) and near the minimum detection limit in all other cell lines. The single agent activity of ZD1839 was highest in cell line A431. In the other cell lines, it was lower and appeared to be independent of EGF-receptor expression levels. The relative antitumor activity (RAA) was low (RAA = 1). Combined exposure to oxaliplatin and ZD1839 (IC30) resulted in significant synergy in 4 out of 6 colorectal cancer (CRC) cell lines and significant antagonism in 4 out of 6 non-colorectal cancer cell lines. Continuous exposure to ZD1839 (IC30) induced a marked G1-phase arrest and dephosphorylation of EGF-receptor in A431, whereas no significant cell cycle perturbation could be detected in the low-expression cell lines. Other factors than cell cycle perturbation seem to determine the mode of drug interaction between oxaliplatin and ZD1839. Based on RAA, the single agent activity of ZD1839 in the investigated cell line panel appeared to be low. Combined exposure to ZD1839 and oxaliplatin exerted synergy in colorectal cancer cell lines, warranting further evaluation in this type of cancer. However, based on the observed antagonism in non-colorectal cancer cell lines, combined treatment with ZD1839 and oxaliplatin is not recommended for other types of cancer. Further research is necessary to identify factors which determine the nature of drug interaction in different tumor types including CRC and lung cancer.
Article
Anecdotal clinical reports denote first tissue engineering applications entering medical practice. Currently it is still unknown, if these new types of implants will tolerate the specific needs in cancer patients undergoing postoperative chemo- and radiotherapy. We implemented a radiotherapy protocol (cumulative dosis 40 Gy) on generated human bioartificial fibromuscular tissues in vitro. We monitored tissue vitality during radiotherapy and tissue recovery (8 weeks follow up period) applying histological methods. The biopsy procedure and seeding techniques yielded a viable 3 dimensional bioartificial human tissue. Radiation resulted in immediate devitalization without destroying tissue integrity. The bioartificial tissue recovered entirely in vitro within 6 weeks. Bioartificial human implants appear applicable for surgical reconstruction in oncologic patients potentially facing postoperative radiotherapy.