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Modulation of tracer accumulation in malignant tumors: gene expression, gene transfer, and phage display.

Assessment of gene function following the completion of human genome sequencing may be done using radionuclide imaging procedures. These procedures are needed for the evaluation of genetically manipulated animals or new designed biomolecules which requires a thorough understanding of physiology, biochemistry and pharmacology. The experimental approaches will involve many new technologies including in vivo imaging with SPECT and PET. Nuclear medicine procedures may be applied for the determination of gene function and regulation using established and new tracers or using in vivo reporter genes such as genes encoding enzymes, receptors, antigens or transporters. Visualization of in vivo reporter gene expression can be done using radiolabeled substrates, antibodies or ligands. Combinations of specific promoters and in vivo reporter genes may deliver information about the regulation of the corresponding genes. Furthermore, protein-protein interactions and activation of signal transduction pathways may be visualized non-invasively. The role of radiolabeled antisense molecules for the analysis of mRNA content has to be investigated. However, possible applications are therapeutic intervention using triplex oligonucleotides with therapeutic isotopes which can be brought near to specific DNA sequences to induce DNA strand breaks at selected loci. Imaging of labeled siRNA's makes sense if these are used for therapeutic purposes in order to assess the delivery of these new drugs to their target tissue. Finally, new biomolecules will be developed by bioengineering methods which may be used for isotope-based diagnosis and treatment of disease.

Animals↗

Tissue engineering using adult stem cells.

Patients with a variety of diseases may be treated with transplanted tissues and organs. However, there is a shortage of donor tissues and organs, which is worsening yearly because of the aging population. Scientists in the field of 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 tissues. The stem cell field is also advancing rapidly, opening new options for cellular therapy and tissue engineering. The use of adult stem cells for tissue engineering applications is promising. This chapter discusses applications of these new technologies for the engineering of tissues and organs. The first part provides an overview of regenerative medicine and tissue engineering techniques; the second highlights different adult stem cell populations used for tissue regeneration.

Adult Stem Cells↗

Tissue engineering using human embryonic stem cells.

The possibility of using stem cells for tissue engineering has greatly encouraged scientists to design new platforms in the field of regenerative and reconstructive medicine. Stem cells have the ability to rejuvenate and repair damaged tissues and can be derived from both embryonic and adult sources. Among cell types suggested as a cell source for tissue engineering (TE), human embryonic stem cells (hESCs) are one of the most promising candidates. Isolated from the inner cell mass of preimplantation stage blastocysts, they possess the ability to differentiate into practically all adult cell types. In addition, their unlimited self-renewal capacity enables the generation of sufficient amount of cells for cell-based TE applications. Yet, several important challenges are to be addressed, such as the isolation of the desired cell type and gaining control over its differentiation and proliferation. Ultimately, combing scaffolding and bioactive stimuli, newly designed bioengineered constructs, could be assembled and applied to various clinical applications. Here we define the culture conditions for the derivation of connective tissue lineage progenitors, design strategies, and highlight the special considerations when using hESCs for TE applications.

Cell Culture Techniques↗

The surgical management of facial nerve injury.

Treatment of facial nerve injuries depends upon a detailed understanding of its anatomic course, accurate clinical examination, and timely and appropriate diagnostic studies. Reconstruction depends upon the extent of injury, the availability of the proximal stump. and the time since injury and duration of muscle denervation. Although no alternative is perfect, these techniques, in combination with static and ancillary procedures. can protect the eye, prevent drooling, restore the smile, and improve facial symmetry. New techniques (including single-stage free tissue transfers and bioengineered nerve grafts), further research on the characteristics of the facial musculature, and methods of preserving the neuromuscular junction will undoubtedly manifest themselves as further refinements of established surgical techniques.

Acute Disease↗

Clinical applications of tissue engineered constructs.

The reconstruction of soft tissue defects poses a challenge for plastic surgeons and tissue engineers. The construction of a biologically, functionally, and cosmetically successful replacement part will involve the combination of a composite that contains endoderm, mesoderm, and ectoderm. It will be active in immune surveillance and function. It must be durable to withstand the stress and strain encountered by the skin. Such a composite will involve the use of bone, cartilage, muscle, blood vessels, nerves, connective tissue, dermis, and epidermis. Fortunately, many of these tissues are among the best studied by tissue engineers. The future of this field will likely involve to some degree the co-mingling of current reconstructive modalities, including the techniques of prefabrication and pre-lamination, with more aggressive and successful tissue engineering technology and the rapidly developing science of stem cell biology. Tissues synthesized in vitro with better structure, color, and texture can be pre-laminated to a site that has already been prefabricated. Prefabrication of a bio-absorbable matrix can create a well perfused scaffold onto which larger subunits can be prelaminated. The future of this field of endeavor is exciting, and, with further research, experience, and interdisciplinary collaboration, bioengineered tissue constructs will become a reality.

Burns↗

Progress in adipose tissue construct development.

Although the field of tissue engineering has been the focus of a great deal of promise and study, only recently has significant attention been given to the engineering of soft tissues. The applicability of an engineered adipose construct as a basic science model and a reconstructive tool is unquestioned; yet, there have been limitations in previous work, specifically issues of construct size and maintenance over time. This article briefly overviews the pivotal factors necessary for adipocyte growth and differentiation, optimal scaffolds for the engineering of soft tissues, and a means of providing vascular support for these highly demanding cells. Clinical science and bioengineering concepts that may provide the foundation toward the successful in vivo engineering of an adipose tissue construct that maintains its complex three-dimensional shape over time are critically reviewed.

Adipocytes↗

Tissue engineering in dentistry.

Advances in tissue engineering provide an increased level of understanding of the mechanical and chemical stimuli that regulate tissue responses. Oral tissue engineering can be applied to recreate missing osseous or dental structures or correct orofacial deformities, changing the patient's smile, midfacial height, and the soft tissue drape. Biomechanical principles can also be applied to tissue engineering to enhance the bone/tooth or bone/implant functionality and long-term stability. Advancements are also being achieved in the area of biomimetics that will allow the creation of new biologic replacements for missing oral structures. The opportunity for bioengineering to charter the course of tooth regeneration is an exciting prospect and will improve the quality of life for patients for decades to come.

Biomechanical Phenomena↗

Tissue engineered fetal skin constructs for paediatric burns.

Autologous skin-grafting is the gold standard for treatment of deep second and third degree burns. Available bioengineered skin products also necessitate this two-step surgical procedure. Therefore, we developed fetal skin constructs to improve healing of such degree burns. A bank of fetal skin cells was developed from one organ donation (4 cm2 of skin allowing the preparation of several million three-dimensional skin constructs, 9x12 cm, on native horse collagen). Successive fetal constructs were applied to eight patients at every change of dressing during 1-3 weeks in an outpatient setting. Complete closure was rapid (mean 15.3 days [SD 5.5]) with little hypertrophy of new skin and no retraction seen. This simple technique provided complete treatment without auto-grafting, showing that fetal skin cells might have great potential to treat burns and eventually acute and chronic wounds of other types.

Burns↗

Behavior and properties of neat and filled gelatins.

The purpose of this study was to determine the mechanical properties and the atomistic structures of various gelatin hydrogels as a preliminary to using these in bioengineering applications. The hydrogels were investigated as neat materials and as particulate-reinforced composites both in the as-formed state and following cross-linking with formaldehyde and/or glutaraldehyde. The compressive modulus obtained using alumina particulates as the reinforcement was found to be enhanced significantly more than expected on the basis of considering a gel matrix to be similar to a thermoplastic one. From the electron paramagnetic resonance of a Cu(2+) probe ion implanted in these materials it was determined that the variation in the compressive moduli with Bloom indices results from a gradation in the relative weightings of two discrete coordination configurations. The cross-linking led to different coordinations following the formaldehyde vs. the glutaraldehyde treatments.

Aluminum Oxide↗

Silk-based biomaterials.

Silk from the silkworm, Bombyx mori, has been used as biomedical suture material for centuries. The unique mechanical properties of these fibers provided important clinical repair options for many applications. During the past 20 years, some biocompatibility problems have been reported for silkworm silk; however, contamination from residual sericin (glue-like proteins) was the likely cause. More recent studies with well-defined silkworm silk fibers and films suggest that the core silk fibroin fibers exhibit comparable biocompatibility in vitro and in vivo with other commonly used biomaterials such as polylactic acid and collagen. Furthermore, the unique mechanical properties of the silk fibers, the diversity of side chain chemistries for 'decoration' with growth and adhesion factors, and the ability to genetically tailor the protein provide additional rationale for the exploration of this family of fibrous proteins for biomaterial applications. For example, in designing scaffolds for tissue engineering these properties are particularly relevant and recent results with bone and ligament formation in vitro support the potential role for this biomaterial in future applications. To date, studies with silks to address biomaterial and matrix scaffold needs have focused on silkworm silk. With the diversity of silk-like fibrous proteins from spiders and insects, a range of native or bioengineered variants can be expected for application to a diverse set of clinical needs.

Animals↗

Animal models for intestinal tissue engineering.

Although total parenteral nutrition prevents patients with short bowel syndrome from dying of starvation, having short bowel remains a severely debilitating condition. The best current treatment for inadequate absorptive surface area is through intestinal transplantation. However, this therapy is associated with significant morbidity and patients suffer from consequences of long-term immunosuppression. Additionally, the numbers of organs are limited. A new frontier in medicine is the field of tissue engineering. We will review the progress of intestinal bioengineering with a focus on the use of animal models. Investigators initially used autologous tissue as a patch to study intestinal regeneration. Subsequent studies focused on the use of absorbable biomaterials as a patch for tissue ingrowth. The most novel methodology consists of seeding a resorbable scaffold and implanting this construct to observe the regeneration of neointestine. Successful creation of esophagus, stomach, small bowel and colon has been demonstrated. Although these studies are preliminary, the results suggest that tissue-engineered intestine will become a real therapeutic option in the not too distant future for patients with inadequate intestinal tissue.

Absorbable Implants↗

Friction in hip prostheses.

Although the reduction of frictional torques was the driving force behind the design of the Charnley prosthesis, later concerns about wear and subsequent loosening of this and other hip replacements have dominated debate within the bioengineering community. To stimulate discussion on the role of friction in loosening, a review of the frictional characteristics of different prostheses was undertaken. The use of simple laboratory screening-type machines in the frictional assessment of different material combinations is discussed together with experiments performed on single axis simulators using both conventional and experimental prostheses. In particular, recent developments in the use of soft layer components are highlighted. Further, the possible link between excessively high frictional torques and loosening is discussed in the light of current results obtained from explanted prostheses.

Alloys↗

Acceptability of bio-engineered vaccines.

For hundreds of years bacterial and viral vaccines have been-in a way-bioengineered and were generally well received by the public, the authorities, and the medical profession. Today, additional tools, e.g. molecular biology, enable new approaches to the development of better and safer products. Various vaccines derived from gene technology have now been licensed for commercial use and are acknowledged within the scientific community. Acceptance by the public and the politicians is, however, negatively influenced by the discussions encompassing gene manipulation in man and animals, transgenic plant, and "novel food". Lack of information leads to confusion and fear. Concurrently, the absence of spectacular and life-threatening epidemics limits the perceived value of immune prophylaxis and its benefits. Scientists in institutes and industry are in a position to stimulate acceptability of bio-engineered vaccines by following some simple rule: (1) adherence to the principles of safety; (2) establishment of analytical and control methods; (3) well functioning regulatory and reporting systems; (4) demonstration of usefulness and economic benefits; (5) open communication; and (6) correct and prudent wording.

Biotechnology↗

Hard tissue remodeling using biofabricated coralline biomaterials.

Biotechnical and biomedical approaches were combined in an attempt to identify potential uses of biofabricated marine carbonate materials in biomedical applications, particularly as biomatrices for remodeling bone and cartilage tissue. After grafting, it is desirable for bone ingrowth to proceed as quickly as possible because the strength of the implanted region depends on a good mechanical bond forming between the implant and surrounding regions in the body. Ingrowth can take place as a result of growth of tissue and cells into the implanted porous material, or it may be promoted by transplanting cells seeded onto such a material. The rate at which ingrowth occurs is dependent on many factors, including pore size and the interconnectivity of the implanted structure. In vivo graftings into osteochondral defects demonstrated that our biofabricated porous material is highly biocompatible with cartilage and bone tissue. The biofabricated matrix was well incorporated into the biphasic osteochondral area. Resorption was followed by bone and cartilage formation, and after 4 months, the biomaterial had been replaced by new tissue. Ossification was induced and enhanced without introduction of additional factors. We believe that this is the first time that such biofabricated materials have been used for biomedical purposes. In face of the obvious environmental disadvantages of harvesting from limited natural resources, we propose the use of bioengineered coralline and other materials such as those cultured by our group under field and laboratory conditions as a possible biomatrix for hard tissue remodeling.

Animals↗

An alternative method for the analysis of neuron passive electrical data which uses integrals of voltage transients.

The traditional method for analyzing passive electrical data from neurons when specific morphological data are unavailable consists of decomposing the voltage response of the cell into a series of exponential functions (the peeling method) and substituting the time constants of these exponential functions into equations derived from cable theory (Rall W, Core conductor theory and cable properties of neurons. In: Handbook of Physiology. The Nervous System. Cellular Biology of Neurons. Bethesda, MD. Am Physiol Soc. Section 1, Part 1, 1977;1(3):39-97). In the present report, an alternative method is examined for analyzing these kinds of data, the integrals of transients method (Eisenberg RS, Mathias RT. Structural analysis of electrical properties of cells and tissues. CRC Critical Reviews in Bioengineering 1980;4:203-232). The integrals required are easily obtained from input resistance data and any theoretical model that is appropriate for the neurons under study can be used, provided that the impedance function can be determined. In order to demonstrate this alternative method, a simple 3-compartment model with both dendritic taper and somatic shunt is used to model data obtained from fast-type alpha-motoneurons in the spinal cord of the cat. These results are compared with results obtained using the traditional peeling method. This comparison indicates that passive electrical data from fast-type motoneurons are best analyzed using a theoretical model that includes both dendritic taper and somatic shunt. Furthermore, our results show that the integrals of transients method can facilitate this analysis.

Animals↗

Mouse feeding behavior: ethology, regulatory mechanisms and utility for mutant phenotyping.

Ingestive behaviors, feeding and drinking, constitute unconditioned, obligatory functions that are tightly regulated in the rodent according to demands of the external and internal milieu. Dependent measures of food intake have been used extensively in rats to infer the identity and function of neurochemical pathways, which mediate energy balance. A recent interest in application of appetitive measures in mice can be attributed jointly to the discovery of novel markers of energy balance in genetically obese mice as well as systematic targeting of known feeding regulatory pathways in bioengineered mutant mice. Accordingly, this review will attempt to provide the reader interested in behavioral phenotyping of knockout or transgenic mice with information regarding the ethology of mouse eating behavior, known mechanisms of appetitive regulation and examples of successes and pitfalls encountered when studying food intake in mutant mice.

Animals↗

Vasoactive intestinal peptide supports induced migration of human keratinocytes and their colonization of an artificial polyurethane matrix.

We investigated the effect of the neuropeptides vasoactive intestinal peptide (VIP), (D-Phe2)-VIP, (Lys-Pro-Arg-Tyr)-VIP and the VIP fragment (1-12) on induced migration and colonization in vitro. In confluent keratinocyte cultures "wounded" with a razor blade, the VIP-treated samples disclosed a more rapid migration from the wound margins into the wound bed, starting within the first 4 h. Almost 80% of the wounded area was covered within 24 h. In contrast, VIP-derivatives were not significantly different from controls, covering 10 to 18% of the wounded area (p < 0.02). Colonization has been assessed with an artificial non-toxic polyurethane matrix. In controls, we were able to observe migration of keratinocytes on the matrix within the first 24 h. The cells, however, were not able to migrate further and to survive. After 48 h, VIP-treated cultures showed a complete colonization of the matrix by keratinocytes vs. less than 10% of the total area in controls (p < 0.001). The induction of migration and of colonization was VIP-dose-dependent. The data indicate that induced migration is stimulated by VIP, when the N-terminal ending is intact, but loss of the C-terminus abrogates both migration and colonization. Our investigations have implications for wound healing but also for bioengineering of skin.

Cell Movement↗

Protein engineering of bacterial alpha-amylases.

alpha-Amylases constitute a very diverse family of glycosyl hydrolases that cleave alpha1-->4 linkages in amylose and related polymers. Recent structural and mutagenic studies of archeael, mammalian and bacterial alpha-amylases have resulted in a wealth of information on the catalytic mechanism and on the structural features of this enzyme class. Because of their high thermo-stability, the Bacillus alpha-amylases have found widespread use in industrial processes, and much attention has been devoted to optimising these enzymes for the very harsh conditions encountered there. Stability has been a major area of focus in this respect, and several remarkably stable bacterial alpha-amylases have been produced by bioengineering techniques. Protein engineering studies of pH-activity profiles and of substrate specificities have also been initiated, although without much success. In the coming years it is likely, however, that the focus of alpha-amylase engineering will shift from engineering stability to these new areas.

Bacillus↗