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PubMed2006

Chloroplast genetic engineering via organogenesis or somatic embryogenesis.

Chloroplast genetic engineering offers a number of unique advantages, including high-level transgene expression, multigene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer desired agronomic traits or express high levels of vaccine antigens and biopharmaceuticals. Despite such significant progress, this technology has not been extended to other important plant species. For example, Arabidopsis may be an ideal model system for chloroplast functional genomics. The employment of chloroplast transformation technology in Arabidopsis has been hampered by the lack of an efficient and reproducible protocol that provides fertile chloroplast transgenic plants. Transformation of the Arabidopsis chloroplast genome was achieved via organogenesis but the efficiency was at least a 100-fold lower than in tobacco and had the drawback of polyploidy in the leaf tissue that resulted in sterile transgenic plants. This problem can be overcome by adapting procedures that are now available to regenerate plants from both diploid and tetraploid explants via callus. In addition, it is feasible to regenerate Arabidopsis via somatic embryogenesis. Recent breakthroughs in highly efficient plastid transformation of recalcitrant crops such as cotton and soybean have opened the possibility of engineering Arabidopsis plastid genome via somatic embryogenesis. Therefore, protocols of recent improvements in tissue culture, DNA delivery, and the novel vector designs are provided here in order to achieve highly efficient plastid transformation in Arabidopsis.

Arabidopsis↗
PubMed2000

The impact of tissue engineering on dentistry.

BACKGROUND: Tissue engineering is a novel and highly exciting field of research that aims to repair damaged tissues as well as create replacement (bioartificial) organs. OVERVIEW: The authors provide a general review of the principles underlying key tissue engineering strategies, as well as the typical components used. Several examples of preclinical and clinical progress are presented. These include passive approaches, such as dental implants, and inductive approaches that activate cells with specific molecular signals. PRACTICE IMPLICATIONS: Tissue engineering will have a considerable effect on dental practice during the next 25 years. The greatest effects will likely be related to the repair and replacement of mineralized tissues, the promotion of oral wound healing and the use of gene transfer adjunctively.

Animals↗
PubMed2004

[Electrospun poly-l-lactide nanofibres as scaffolds for tissue engineering].

Tissue engineering is a promising tool for treating structural and functional defects in bone and cartilage. To provide optimal conditions for three-dimensional cell growth the use of a scaffold is necessary. The aim of the study was to test the potential application of an electrospun poly (l-lactide)-nanostructured scaffold as a matrix for tissue engineering. Matrices were seeded with human osteosarcoma MG-63 cells and cultivated for 14 days. Cells showed a clear preference for growth along the nanofibres, and demonstrated no signs of degeneration or apoptosis. The fine structure of electrospun nanofibres makes them an ideal scaffold for tissue engineering, in particular for cartilage repair. They can be "doped" with growth factors, medications, etc., and are both biocompatible and biodegradable.

Apoptosis↗
PubMed2002

Tissue-engineered skin substitutes.

The last two years have seen new tissue-engineered skin substitutes come onto the market and begin to resolve the various roles to which each is best suited. It is becoming evident that some of the very expensive cell-based products have cost-benefit advantage despite their high price and are valuable within the restricted applications for which they are intended. The use of skin substitutes for testing purposes has extended from epidermal keratinocytes to other integumentary epithelia and into preparations containing multiple cell types in which reactions resulting from paracrine interactions can be examined. Challenges remain in the application of gene therapy techniques to skin substitutes, both the control of transgene expression and in the selection of suitable genes to transfect. A coming challenge is the production of tissue-engineered products without the use of animal products other than human cells. A challenge that may be diminishing is the importance of acute rejection of allogeneic tissue-engineered skin substitutes.

Animals↗
PubMed2006

Genetically engineered underdominance for manipulation of pest populations: a deterministic model.

We theoretically investigate the potential for introgressing a desired engineered gene into a pest population by linking the desired gene to DNA constructs that exhibit underdominance properties. Our deterministic model includes two independently segregating engineered constructs that both carry a lethal gene, but suppress each other. Only genotypes containing both or neither construct are viable. Both constructs also carry the desired gene with an independent regulatory mechanism. We examine the minimal number of individuals of an engineered strain that must be released into a natural population to successfully introgress the desired gene. We compare results for strains carrying single and multiple insertions of the constructs. When there are no fitness costs associated with the inserted constructs (when the lethal sequences are not expressed), the number of individuals that must be released decreases as the number of insertions in the genome of the released strain increases. As fitness costs increase, the number of individuals that must be released increases at a greater rate for release strains with more insertions. Under specific conditions this results in the strain with only a single insertion of each construct being the most efficient for introgressing the desired gene. We discuss practical implications of our findings.

Animals↗
PubMed1990

[Mental health in software engineers. II. Classification of occupational stressors, and relationship between occupational stressors and psychiatric disorders].

Occupational stressors and psychiatric disorders were studied in a sample of 101 male software engineers. Psychiatric diagnosis was based on DSM-III (Diagnostic and Statistical Manual of Mental Disorders, Third Edition). The results can be summarized as follows: 1) More than 30% of the subjects were not satisfied with their work. 2) Occupational stressors ascertained in the interview could be classified into "quantity of job," "quality of job," "role in organization," "relations with others," "work conditions," "career development," "reward," "organizational structure and climate," "low social support," and "lack of decision making." "quality of job" (35%), "quantity of job" (30%), and "role in organization" (33%) were the most common stressors for the subjects. 3) Among the occupational stressors, only "quantity of job" was significantly related to psychiatric disorders (p less than 0.05). The results suggest that many software engineers are not satisfied with their work and that "quantity of job" has the greatest influence on their mental health. Some measures should be taken to avoid potential health hazards, for the research findings indicate a relationship between occupational stressors and mental health in software engineers.

Adult↗
PubMed2006

Growth factor-delivery systems for tissue engineering: a materials perspective.

The transplantation of organs, their surgical reconstruction or implantation of synthetic devices that can perform the function of organs, are the currently available methods for treating loss of tissue/organs in humans. However, the limitations associated with these techniques have led to the development of tissue engineering. One of the primary goals of tissue engineering is to provide growth factor delivery systems that can induce desired cell responses both in vitro and in vivo, in order to cause accelerated tissue regeneration. To make growth factors a more therapeutically viable alternative for the treatment of chronic degenerative diseases, a wide range of natural and synthetic materials have been employed as vehicles for their controlled delivery. The choice of material and design of the carrier device influence the mode of immobilization of growth factors on the scaffolds and their local/systemic administration. From a tissue engineer's perspective, materials could be used for designing scaffolds as well as for delivering single or multiple growth factors. Therefore, this review discusses growth factor delivery systems, with particular reference to carrier-based growth factor delivery systems with a focus on materials.

Animals↗
PubMed2006

Progress in the development of a corneal replacement: keratoprostheses and tissue-engineered corneas.

Rapid progress has been made in the past 5 years in the development of corneal replacements. Traditionally they are divided into two categories, keratoprostheses and tissue-engineered corneal equivalents, as replacement tissues are increasingly in demand worldwide. There are currently several different keratoprosthesis models in clinical use around the world. The most popular and most widely publicized is the AlphaCor model, which has enjoyed significant clinical success. However, improvements remain to be made, and the aim of most of the current research is to better understand the interactions between a synthetic material and the surrounding biology on a more fundamental level. This improved understanding will no doubt lead to improvements in current models and to the development of new models in the near future. While tissue-engineered corneal equivalents have been under investigation for considerably less time, there is growing evidence to suggest that a tissue-engineered corneal equivalent comprised of primarily natural materials will exist in the not too distant future. Research groups have reported strong in vitro and in vivo results. The strength of the collagen matrix and its ability to support cell infiltration have been the primary avenues of research. Various collagen crosslinking techniques have been used. Infiltration of three major cells of the cornea has been observed. Most importantly, the ability of these materials to support nerve ingrowth has been demonstrated. While challenges remain with both types of corneal replacements, the considerable progress in the recent past suggests that reliable implants for the treatment of a variety of corneal diseases will be available. This review will provide an overview of recent results, and will provide insight into the future of research on corneal replacements.

Animals↗
PubMed2002

Scaffolds for tissue engineering of cartilage.

Articular cartilage lesions resulting from trauma or degenerative diseases are commonly encountered clinical problems. It is well-established that adult articular cartilage has limited regenerative capacity, and, although numerous treatment protocols are currently employed clinically, few approaches exist that are capable of consistently restoring long-term function to damaged articular cartilage. Tissue engineering strategies that focus on the use of three-dimensional scaffolds for repairing articular cartilage lesions offer many advantages over current treatment strategies. Appropriate design of biodegradable scaffold conduits (either preformed or injectable) allow for the delivery of reparative cells bioactive factors, or gene factors to the defect site in an organized manner. This review seeks to highlight pertinent design considerations and limitations related to the development, material selection, and processing of scaffolds for articular cartilage tissue engineering, evidenced over the last decade. In particular, considerations for novel repair strategies that use scaffolds in combination with controlled release of bioactive factors or gene therapy are discussed, as are scaffold criteria related to mechanical stimulation of cell-seeded constructs. Furthermore, the subsequent impact of current and future aspects of these multidisciplinary scaffold-based approaches related to in vitro and in vivo cartilage tissue engineering are reported herein.

Animals↗
PubMed2006

Encapsulation cell therapy for mucopolysaccharidosis type VII using genetically engineered immortalized human amniotic epithelial cells.

Mucopolysaccharidosis type VII (MPSVII) is a lysosomal storage disease resulted from a deficiency of the enzyme beta-glucuronidase (GUSB), which is necessary for degradation of glycosaminoglycans (GAGs). The deficiency of GUSB causes progressive accumulation of GAGs and subsequent lysosomal distension in multiple tissues, including the central nervous system (CNS). In murine experiments, bone marrow transplant, enzyme replacement, viral vectors, and genetically modified cells were successfully used for correction of the visceral accumulation of GAGs, but little improvement was seen in the brain, because these therapeutic agents cannot cross the blood-brain barrier (BBB). Although direct intracerebral injection of GUSB-encoding viral vectors has been developed to bypass the BBB, the possibility of tumor formation and the toxicity of over-expressed GUSB have been reported. In this study, we generated immortalized human amniotic epithelial (IHAE) cells to maintain the effect of implantation, and encapsulated these cells to prevent harmful immunological response and tumor formation and to regulate the level of GUSB expression within the host. Moreover, we generated IHAE cells that over-express and secrete human GUSB following transduction with an adenoviral vector encoding human GUSB. Therapeutic efficacy for MPSVII was evaluated in and ex vivo experiments using these encapsulated genetically engineered GUSB-encoding IHAE cells. We confirmed that encapsulated genetically engineered IHAE cells could secrete significant amounts of GUSB outside the capsule in vitro and into the cerebral parenchyma of C3H mice seven days after the capsule implantation. Thus, encapsulation cell therapy using genetically engineered IHAE cells is an effective armamentarium for the treatment of MPSVII.

Amnion↗
PubMed2006

Erythropoietin delivery by genetically engineered bone marrow stromal cells for correction of anemia in mice with chronic renal failure.

The goal of this research was to develop a strategy to couple stem cell and gene therapy for in vivo delivery of erythropoietin (Epo) for treatment of anemia of ESRD. It was shown previously that autologous bone marrow stromal cells (MSCs) can be genetically engineered to secrete pharmacologic amounts of Epo in normal mice. Therefore, whether anemia in mice with mild to moderate chronic renal failure (CRF) can be improved with Epo gene-modified MSCs (Epo+MSCs) within a subcutaneous implant was examined. A cohort of C57BL/6 mice were rendered anemic by right kidney electrocoagulation and left nephrectomy. In these CRF mice, the hematocrit (Hct) dropped from a prenephrectomy baseline of approximately 55% to 40% after induction of renal failure. MSCs from C57BL/6 donor mice were genetically engineered to secrete murine Epo at a rate of 3 to 4 units of Epo/10(6) cells per 24 h, embedded in a collagen-based matrix, and implanted subcutaneously in anemic CRF mice. It was observed that Hct increased after administration of Epo+MSCs, according to cell dose. Implants of 3 million Epo+MSCs per mouse had no effect on Hct, whereas 10 million led to a supraphysiologic effect. The Hct of CRF mice that received 4.5 or 7.5 million Epo+MSCs rose to a peak 54+/-4.0 or 63+/-5.5%, respectively, at 3 wk after implantation and remained above 48 or 54% for >19 wk. Moreover, mice that had CRF and received Epo+MSCs showed significantly greater swimming exercise capacity. In conclusion, these results demonstrate that subcutaneous implantation of Epo-secreting genetically engineered MSCs can correct anemia that occurs in a murine model of CRF.

Anemia↗
PubMed1998

Isolation of human osteoblast-like cells and in vitro amplification for tissue engineering.

As the field of dental implants continues to grow at a rapid rate so does our quest to find new techniques to enhance bone grafting. Tissue engineering is an exciting new technique in bone grafting. Therefore, the purposes of this study were to develop a simple, reproducible method to isolate human osteoblast-like cells (HOBs) and to evaluate in vitro cell proliferation within 2 different 3-dimensional (3-D) constructs targeted for tissue engineering applications. Ultimately, HOBs that have been amplified within 3-D constructs may be employed for bone regeneration techniques, such as onlay and sinus grafting prior to implant placement. Our cell isolation protocol employed human fetal calvaria tissue sequentially digested with trypsin and collagenase. The HOB cells from only the third and fourth digests were obtained, cultured and evaluated within the constructs. An osteoblast-like phenotype was in part verified for these HOB cells by demonstrating a significantly higher alkaline phosphatase activity than for human gingival fibroblasts, and a comparable level to the osteoblast cell line MG-63. The HOB cells were cultured within either poly (D,L-lactide) (PLA) or a fused fiber ceramic and evaluated for the ability to support in vitro HOB amplification. HOB proliferation was validated by scanning electron microscopy, identifying cells throughout the 3-D constructs. Continuous cell viability was demonstrated for the duration of the 33-day evaluation period and the extent of cell amplification reached approximately 20 times the seeding density. The in vitro amplification results further indicate that tissue engineering strategies with either the PLA or fused fiber construct may be suitable for bone regeneration therapy for dental implants.

Biomedical Engineering↗
PubMed2003

In vivo somatic cell gene transfer of an engineered Noggin mutein prevents BMP4-induced heterotopic ossification.

BACKGROUND: The formation of the skeleton requires inductive signals that are balanced with their antagonists in a highly regulated negative feedback system. Inappropriate or excessive expression of BMPs (bone morphogenetic proteins) or their antagonists results in genetic disorders affecting the skeleton, such as fibrodysplasia ossificans progressiva. BMP signaling mediated through binding to its receptors is a critical step in the induction of abnormal ossification. Therefore, we hypothesized that engineering more effective inhibitors of this BMP-signaling process may lead to the development of therapies for such conditions. METHODS: BMP4-induced heterotopic ossification was used as a model for testing the ability of the BMP antagonist Noggin to block de novo bone formation, either by local or systemic delivery. Since Noggin naturally acts locally, a Noggin mutein, hNOGDeltaB2, was engineered and was shown to circulate systemically, and its ability to block heterotopic ossification was tested in a mouse model with use of adenovirus-mediated somatic cell gene transfer. RESULTS: A mouse model of BMP4-induced heterotopic ossification was developed. Local delivery of wild-type NOG inhibited heterotopic ossification, but systemic administration was ineffective. In contrast, systemic delivery of the adenovirus encoding hNOGDeltaB2 resulted in systemic levels that persisted for more than two weeks and were sufficient to block BMP4-induced heterotopic ossification. CONCLUSIONS: BMP4-induced heterotopic ossification can be prevented in vivo either by local delivery of wild-type Noggin or after somatic cell gene transfer of a Noggin mutein, hNOGDeltaB2. Furthermore, the data in the present study provide proof of concept that a naturally occurring factor can be engineered for systemic delivery toward a desirable pharmacological outcome. CLINICAL RELEVANCE: Blocking bone formation is clinically relevant to disorders of heterotopic ossification in humans, such as fibrodysplasia ossificans progressiva. Furthermore, development of BMP antagonists as therapeutic agents may provide modalities for the treatment of other pathologic conditions that arise from aberrant expression of BMPs and/or from a lack of their antagonists.

Adenoviridae↗
PubMed2000

Genetically engineered monoclonal antibodies for direct anti-neoplastic treatment and cancer cell specific delivery of chemotherapeutic agents.

Classical therapeutic modalities such as surgery, radiation, and chemotherapy not only fail to cure the great majority of malignant tumors, but their employment often leads to severe and debilitating side effects. The severe cancer related morbidity is also in direct correlation with the use of x-radiation and chemotherapy, making them less than ideal forms of therapy. The development of hybridoma technology and the advances in monoclonal antibody (MoAB) production have revitalized the initial concept of Ehrlich concerning the existence of cancer cell-targeted, specific "magic bullets". Entirely new approaches to cancer therapy that are neoplastic cell-directed, and specifically lethal to malignant cells and less toxic to normal tissues are being observed and developed, adhering to the old prayer: "Destroy the diseased tissues, preserve the normal." Immunotherapy as a fourth modality of cancer therapy has already been developed and proven to be quite effective. Strategies for the employment of antibodies for anti-cancer immunotherapy include: 1) Immune reaction directed destruction of cancer cells; 2) Interference with the growth and differentiation of malignant cells; 3) Antigen epitope directed transport of anti-cancer agents to malignant cells; 4) Anti-idiotype vaccines; and 5) Development of engineered (humanized) mouse monoclonals for anti-cancer therapy. In addition, a variety of different agents (e.g. toxins, radionuclides, chemotherapeutic drugs) have been conjugated to mouse and human MoABs for selective delivery to cancer cells. Preclinical observations in athymic, nude mice using xenografted human cancers and mouse, anti-human MoABs were more than impressive and have lead to the development of clinical trials. Phase I studies established the safety of employing immunoconjugates in humans, but the in vivo therapeutic results were less impressive. The clinical use of mouse MoABs in humans is limited due to the development of a foreign anti-globulin immune response by the human host. Genetically engineered chimeric human-mouse MoABs have been developed by replacing the mouse Fc region with the human constant region. Moreover, the framework regions of variable domains of rodent immunoglobulins were also experimentally replaced by their human equivalents. These antibodies can also be designed to have specificities and effector functions determined by researchers, which may not appear in nature. The development of antibodies with two binding ends (bispecific antibodies) provided a great improvement in targeting cancer cells. The existing inadequacies of MoABs in immunotherapy may also be improved by increasing their efficiency with chemical coupling to various agents such as bacterial or plant toxins, radionuclides or cytotoxic drugs. The astonishing immunophenotypic (IP) heterogeneity of neoplastically transformed cells, the different cytotoxic activity associated with the moiety linked to given MoABs, and mostly the impressive genetic modulation capabilities of cancer cells still remain as yet unsolved difficulties in the present immunotherapy of human cancer. In writing this review article, one of our main goals is to encourage further clinical research with the use of genetically engineered rodent MoABs and various immunoconjugates in the treatment of human cancer, as well as the combination of such immunotherapy with the three conventional modalities of therapy. Finally, we propose that MoAB-based immunotherapy be accepted as a conventional form of therapy and employed not only in terminal cancer patients but also, for instance, during and following surgical resection.

Animals↗
PubMed2000

Engineering novel bioactive mini-proteins from small size natural and de novo designed scaffolds.

Mini-proteins, polypeptides containing less than 100 amino acids, such as (animal toxins, protease inhibitors, knottins, zinc fingers, etc.) represent successful structural solutions to the need to express a specific binding activity in different biological contexts. Artificial mini-proteins have also been designed de novo, representing simplified versions of natural folds and containing natural or artificial connectivities. Both systems have been used as structural scaffolds in the engineering of novel binding activities, according to three main approaches: i) incorporation of functional protein epitopes into structurally compatible regions of mini-protein scaffolds; ii) random mutagenesis and functional selection of particular structural regions of mini-protein scaffolds; iii) minimization of protein domains by the use of sequence randomization and functional selection, combined with structural information, in an iterative process. These newly engineered mini-proteins, with specific and high binding affinities within a small size and well-defined three-dimensional structure, represent novel tools in biology, biotechnology and medical sciences. In addition, some of them can also be directly used in therapy or present high potential to serve as drugs. In all cases, they represent precious structural intermediates useful to identify frameworks for peptidomimetic design or directly lead to new small organic structures, representing novel drug candidates. The engineering of novel functional mini-proteins has the potential to become a fundamental step towards the conversion of a protein functional epitope or a flexible peptide lead into a classical pharmaceutical.

Amino Acid Motifs↗
PubMed2004

Glutamate signalling and its potential application to tissue engineering of bone.

Mechanical loading of the skeleton is important for maintenance of adequate bone mass and defined mechanical stimuli are highly osteogenic. The identification of mechanoresponsive signalling molecules in bone may allow osteogenic signals to be mimicked. This approach would be useful in the treatment of bone pathologies where the skeleton is too weak to withstand osteogenic forces and to tissue engineering of bone where the mechanical environment of bone cells is disrupted. Glutamate has been implicated as a mediator of mechanical signalling in bone. Evidence for glutamate signalling in bone, its role in mechanotransduction and potential applications of this pathway to tissue engineering of bone is considered in this review. Glutamate receptors, transporters and proteins that regulate glutamate release, are all expressed in bone cells. Glutamate receptor activation affects both osteoblast and osteoclast phenotypes revealing a potential for therapeutic manipulation of glutamate signalling to enhance bone formation. Glutamate transporters contribute to this system by regulating extracellular glutamate concentrations and acting as glutamate-gated ion channels. Artificial regulation of glutamate receptors or transporters may be used to increase the bone forming capacity of osteoblasts. This novel approach may potentially enhance bone tissue engineering strategies.

Animals↗
PubMed1995

Effects of marital status and position on personality traits in engineers of a computer manufacturing plant.

To investigate the effects of marital status and position on DSM-III-R personality traits, we administered the self-rating questionnaires of the Structured Clinical Interview for DSM-III-R Personality Disorders to 757 male computer engineers, aged 20 to 49 years, at a computer manufacturing plant. The scores for six of eleven personality traits were significantly higher in single computer engineers than in married ones. Also, among the engineers aged 40 to 49, the scores for six personality traits of section-heads were significantly higher than those among persons in the other positions. These findings suggest that "maladaptive" personality traits, defined by the DSM-III-R, differ with marital status and position in a company.

Adult↗
PubMed2004

In vivo engineering of blood vessels.

The inadequacy of conventional synthetic grafts has led to efforts to construct a superior vascular graft. In vivo tissue engineering is one approach to this problem that has been investigated for half a century and enables the construction of autogenous vascular prostheses. Three types of in vivo engineering are explored: remodelling of implanted scaffolds, fibrocollagenous tubes, and the artificial artery generated in the peritoneal cavity. Scaffolds designed to be remodelled may be synthetic or biological and have been remodelled in animal models to form vasoactive neoarteries with arterial morphology. The differences in vascular remodelling ability, particularly spontaneous endothelialisation, between animal models and humans may impair the effectiveness of this approach in the clinic. Fibrocollagenous tubes such as the Sparks Mandril have demonstrated poor performance in the clinic and are prone to aneurysm formation. The artificial artery generated in the peritoneal cavity is a novel addition to the ranks of in vivo engineered vascular prostheses and combines many of the best features of scaffolds designed to be remodelled and fibrocollagenous tubes. However, understanding and manipulating the vascular remodelling process will be the key to producing the ideal arterial prosthesis.

Animals↗