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Tissue engineering: the biophysical background.

Tissue engineering is the construction, repair or replacement of damaged or missing tissue in humans and other animals. This engineering may take place within the animal body or as tissue constructs to be made in a bioreactor for later grafting into the animal. The minimal set of materials for this are the appropriate types of cell. Usually, however, non-living substrata are used as well. These substrata may be nothing more than materials that bulk up any voids in the damaged tissue and provide the mechanical strength that has been lost when the tissue is damaged or removed. They may serve a similar pair of functions in the bioreactor. They can do much more in terms of pattern formation. The orientations and morphology of the cells, the arrangement of intercellular material as it is laid down and the relationships between different cell types in the repairing or construct tissue are all of importance, for these should resemble the correct normal tissue as closely as possible. Most of these requirements are ones involving pattern formation. This review discusses the various ways in which tissue pattern can be engineered chiefly from a biophysical standpoint. Unpatterned cells are effectively not tissue. This engineering includes the use of topography on the substrata, chemical patterning of adhesive and other cues for the cells, mechanical force application to cause cell orientation and appropriate synthetic responses and electrical fields. The review also discusses the methods used to impart the appropriate cues to and through the materials which are often biodegradable polymers. The article gives particular attention to regions of research and practice where the involvement of the physicist or biophysicist is of importance.

Animals↗

Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy.

Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

Adult↗

Tissue-engineered scaffolds are effective alternatives to autografts for bridging peripheral nerve gaps.

The use of autografts for "bridging" peripheral nerve gaps is limited by lack of suitable donor nerve grafts. Using a tissue-engineering approach, we have designed a three-dimensional scaffold that presents laminin 1 (LN-1) and nerve growth factor (NGF) in vivo. Semipermeable polysulfone tubes were used as carriers to introduce the tissue-engineered scaffolds to a 10-mm sciatic nerve gap in adult rats. Two months after implantation, the gross morphology of the regenerated nerve, the success rate of regeneration, and the total number and density of myelinated axons in the tissue-engineered scaffolds matched that observed in autografts. LN-1- and NGF-containing scaffolds performed comparably to autografts when functional measures that include the relative gastrocnemius muscle weight and the sciatic functional index were quantified. Our results demonstrate that tissue-engineered scaffolds match the performance of autografts in an in vivo model of peripheral nerve regeneration, raising the possibility of the scaffolds being used clinically instead of scarce autografts.

Animals↗

Supporting communication in rehabilitation engineering teams.

The objectives of this project were to examine how members of a colocated rehabilitation engineering team communicate during their work and hence deduce the implications of these communications for the design of video-based technologies to support communication among members of a virtual rehabilitation engineering team. Twenty-four assessment clinic sessions conducted by rehabilitation engineering team were recorded on videotape over a period of 3 years. These tapes were analyzed in considerable detail using a schema to identify and classify the talk and actions of the team members. Combining talk and actions with artifacts is a mechanism used by designers to develop ideas and communicate them to others. Speakers rely on actions to support and make their talk lucid. Cooperation based on sharing artifacts is a strength of face-to-face interaction. Participants can experience artifacts and observe others using the artifacts. Tools such as videoconferencing to support virtual rehabilitation teams will have to provide the participants with the ability to see often quite subtle gestures and actions if they are to grasp the meaning of the talk. Increased understanding how a team communicates visually complex data may (1) aid development of next generation videoconferencing equipment to better support distributed designers and rehabilitation engineers and (2) guide development of techniques to enhance the quality of visual data presentation in current videoconferencing systems.

Communication↗

Regression of engineered tumor cells secreting cytokines is related to a shift in host cytokine profile from type 2 to type 1.

The precise role of the endogenous immune response in modulating cancer development remains unclear. In this study, three mouse tumor cell lines were used to elucidate the immune mechanisms for tumor regression versus tumor growth. These cell lines were (1) the poorly immunogenic VKCK cell line and (2) its two derived cell lines VKCK/RM4-tumor necrosis factor-alpha (TNF-alpha) and VKCK/RM4-interferon-gamma (IFN-gamma) engineered to secrete TNF-alpha and IFN-gamma, respectively. Our data showed that VKCK tumors grew aggressively in syngeneic BALB/c mice, and vaccination of irradiated VKCK cells failed to protect the mice from a subsequent challenge with the same tumor. In contrast, engineered VKCK tumor cells lost their tumorigenicity, and vaccination of engineered VKCK cells induced a protective immunity against VKCK cells that was mediated with VKCK-specific CD8+T cells. Susceptible mice developed a Th2-dominant response, whereas resistant mice developed a Th1-dominant response to VKCK. The T cell proliferative response and cytolytic activity against VKCK developed in both resistant and susceptible mice, but in the susceptible mice, these responses were much weaker compared with those in the resistant mice. Our results indicate that regression of tumor cells engineered to secrete cytokines TNF-alpha and IFN-gamma is related to a shift from a host type 2 to a type 1 cytokine profile. Our results further suggest that the failure of unmodified VKCK to generate efficacious T cells is not due to an inability to recognize tumor antigens but, rather, to the nature and magnitude of the antitumor immune response that develops. A better understanding of the mechanisms by which tumor cells modulate the host immune system may result in newer approaches for manipulating host-tumor interactions that favor the development of a protective antitumor immune response.

Animals↗

Analysis of low molecular weight hydrocarbons including 1,3-butadiene in engine exhaust gases using an aluminum oxide porous-layer open-tubular fused-silica column.

A method for the quantitative analysis of individual hydrocarbons in the C1-C8 range emitted in engine exhaust gases is described. The procedure provides base-line or near base-line resolution of C4 components including 1,3-butadiene. With a run time of less than 50 min, the light aromatics (benzene, toluene, ethyl benzene, p- and m-xylene, and o-xylene) are resolved during the same analysis as aliphatic hydrocarbons in the C1-C8 range. It is shown that typical 1,3-butadiene levels in engine exhaust are about 5 ppm at each of two engine conditions. Aromatic hydrocarbon levels show a dependence on engine operating conditions, benzene being about 20 ppm at high speed and about 40 ppm at idle.

Aluminum Oxide↗

Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose.

A significant percentage of eukaryotic proteins contain posttranslational modifications, including glycosylation, which are required for biological function. However, the understanding of the structure-function relationships of N-glycans has lagged significantly due to the microheterogeneity of glycosylation in mammalian produced proteins. Recently we reported on the cellular engineering of yeast to replicate human N-glycosylation for the production of glycoproteins. Here we report the engineering of an artificial glycosylation pathway in Pichia pastoris blocked in dolichol oligosaccharide assembly. The PpALG3 gene encoding Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol mannosyltransferase was deleted in a strain that was previously engineered to produce hybrid GlcNAcMan(5)GlcNAc(2) human N-glycans. Employing this approach, combined with the use of combinatorial genetic libraries, we engineered P. pastoris strains that synthesize complex GlcNAc(2)Man(3)GlcNAc(2) N-glycans with striking homogeneity. Furthermore, through expression of a Golgi-localized fusion protein comprising UDP-glucose 4-epimerase and beta-1,4-galactosyl transferase activities we demonstrate that this structure is a substrate for highly efficient in vivo galactose addition. Taken together, these data demonstrate that the artificial in vivo glycoengineering of yeast represents a major advance in the production of glycoproteins and will emerge as a practical tool to systematically elucidate the structure-function relationship of N-glycans.

Base Sequence↗

Repeat region engineering of Cas13a crRNA enables conformational gating-based autocatalytic CRISPR biosensing.

CrRNA engineering has emerged as a pivotal strategy for extending CRISPR-Cas13a biosensing. However, structural modulation of the direct repeat (DR) region remains exceptionally challenging due to its intricate architecture and the high energetic barrier of the Cas13a-crRNA interface, which is conventionally viewed as a rigid and immutable scaffold. Here, we demonstrate that the DR region is instead a programmable topological element with unexpected structural plasticity. By systematically engineering the DR through sequence insertion and structural splitting, we identified multiple DR variants that retain robust catalytic activity. Crucially, this topological reconfiguration enables Cas13a activity to be precisely gated by unmodified nucleic acid blockers, a level of regulation unattainable with the wild-type crRNA. Building on this flexible modulation, we developed Dre-CRISPR, a DR-engineered platform that couples target-triggered DR restoration to a self-reinforcing autocatalytic loop. This self-amplifying system provides a 2 × 106-fold sensitivity enhancement over nonamplified systems. Furthermore, the Dre-CRISPR platform extends the diagnostic scope of Cas13a to a broader spectrum of analytes, ranging from microRNAs to enzymatic activities and heavy metal ions. Our findings redefine the crRNA scaffold as a versatile signaling node and provide a generalizable framework for developing high-sensitivity, self-amplifying CRISPR biosensors through topology-driven guide RNA engineering.

CRISPR-Associated Proteins↗

Biomechanical factors in tissue engineered meniscal repair.

Damage to the meniscus after trauma or injury is associated with detrimental changes in joint function that can lead to pain, disability, and degenerative joint changes. Recently, tissue engineering strategies for meniscal repair have been suggested including using biocompatible grafts as a substrate for regeneration, and cellular supplementation to promote remodeling and healing. Little is known, however, about the contributions of these novel repair strategies to restoration of normal meniscal function. Biomechanical factors play a role in the design and synthesis of tissue engineered biomaterials and bioreactors, and also are important for evaluating the efficacy of these new strategies for restoring normal meniscal function. In this report, an overview is presented of biomechanical factors that are critical to meniscal function followed by a review of biomechanical considerations for the design and evaluation of tissue engineered strategies for meniscal repair. Recommendations for future study of biomechanical factors in tissue engineered meniscal repair also are provided.

Animals↗

An overview of tissue engineered bone.

Numerous important developments in tissue engineering of new bone during the last 10 years are reviewed. Early efforts to combine cells with biocompatible materials are described and applications of this technology are presented with particular focus on uses in orthopaedics and maxillofacial surgery. Basic principles of tissue engineering focusing on cell biology and materials science as used currently in the field are presented. Finally, future challenges are outlined from the perspective of integrating technologies from medicine, biology, and engineering in hopes of translating tissue engineering to clinical applications.

Animals↗

Intraoral wound closure with tissue-engineered mucosa: new perspectives for urethra reconstruction with buccal mucosa grafts.

In urethra reconstruction, the creation of a new urethra from a free oral mucosa graft is an established surgical technique. The oral mucosa is removed at the same time that the urethra reconstruction procedure is performed. Depending on the size of graft required, the intraoral wound is closed primarily or left to heal secondarily. The latter method limits this technique by leading to scars or strictures, which have a negative impact on the condition of the intraoral soft tissue. Therefore, in this study, a pilot study involving 12 patients, tissue-engineered mucosa was tested for covering intraoral defects to avoid the drawbacks mentioned above. For mucosa tissue-graft engineering, a biopsy sample 2 to 4 mm in diameter was removed from the hard palate approximately 4 weeks before the urethra reconstruction procedure was to be performed. In addition, 30 ml of autogenous serum was extracted from a venous whole-blood sample. The primary cultures were incubated in Dulbecco modified Eagle's medium and nutrient factor F 12 (Gibco Co., Eggenstein, Germany), containing the usual additives and autogenous serum. After a period of 3 weeks, subcultivation was performed to engineer mucosa transplants consisting of several layers of keratinocytes on a support foil. After thorough intraoperative blood coagulation had occurred, the cultured mucosa graft on the carrier foil was applied on the wound surface and fixed by single sutures. Additionally, the cultured mucosa graft was covered for 8 to 10 days by an intraoral dressing, which was also fixed onto the wound surface by single suture loops. It is possible to perform primary intraoral wound closure with tissue-engineered mucosa to cover defect sizes as large as 11.0 x 4.0 cm. This new method provides a better prospect for both urethra reconstruction and the reconstruction of intraoral tissue defects. The number and size of intraoral scars and strictures are diminished. This is of special interest for the reconstruction of the functional unit oral cavity, including soft tissue and cosmetic conditions (e.g., in case of prosthetic rehabilitation). In comparison to primary wound closure with local tissue, the technique presented in this study reduces the severity of postoperative pain and allows faster rehabilitation in patients because of a better wound-healing process. Furthermore, better mobility of intraoral soft tissue structures is achieved.

Adolescent↗

Engineered histones reshape chromatin in human cells.

Histone proteins and their variants have been found to play crucial and specialized roles in chromatin organization and the regulation of downstream gene expression; however, the relationship between histone sequence and its effect on chromatin organization remains poorly understood, limiting our functional understanding of sequence variation between distinct subtypes and across evolution and frustrating efforts to rationally design synthetic histones that can be used to engineer specified cell states. Here, we make the first advance towards engineered histone-driven chromatin organization. By expressing libraries of sequence variants of core histones in human cells, we identify variants that dominantly modulate chromatin structure. We further interrogate variants using a combination of imaging, proteomics, and genomics to reveal both cis and trans-acting mechanisms of effect. Functional screening with transcription factor libraries identifies transcriptional programs that are facilitated by engineered histone expression. Double mutation screens combined with protein language models allow us to learn sequence-to-function patterns and design synthetic histone proteins optimized to drive specific chromatin states. This work establishes a foundation for the high-throughput evaluation and engineering of chromatin-associated proteins and positions histones as tunable nodes for understanding and modulating mesoscale chromatin organization.

Journal Article↗

Quantum four-stroke heat engine: thermodynamic observables in a model with intrinsic friction.

The fundamentals of a quantum heat engine are derived from first principles. The study is based on the equation of motion of a minimum set of operators, which is then used to define the state of the system. The relation between the quantum framework and the thermodynamical observables is examined. A four-stroke heat engine model with a coupled two-level system as a working fluid is used to explore the fundamental relations. In the model used, the internal Hamiltonian does not commute with the external control field, which defines the two adiabatic branches. Heat is transferred to the working fluid by coupling to hot and cold reservoirs under constant field values. Explicit quantum equations of motion for the relevant observables are derived on all branches. The dynamics on the heat transfer constant field branches is solved in closed form. On the adiabats, a general numerical solution is used and compared with a particular analytic solution. These solutions are combined to construct the cycle of operation. The engine is then analyzed in terms of the frequency-entropy and entropy-temperature graphs. The irreversible nature of the engine is the result of finite heat transfer rates and frictionlike behavior due to noncommutability of the internal and external Hamiltonians.

Journal Article↗

Metabolic modeling identifies key constraints on an engineered glycine betaine synthesis pathway in tobacco.

Previous work has shown that tobacco (Nicotiana tabacum) plants engineered to express spinach choline monooxygenase in the chloroplast accumulate very little glycine betaine (GlyBet) unless supplied with choline (Cho). We therefore used metabolic modeling in conjunction with [(14)C]Cho labeling experiments and in vivo (31)P NMR analyses to define the constraints on GlyBet synthesis, and hence the processes likely to require further engineering. The [(14)C]Cho doses used were large enough to markedly perturb Cho and phosphocholine pool sizes, which enabled development and testing of models with rates dynamically responsive to pool sizes, permitting estimation of the kinetic properties of Cho metabolism enzymes and transport systems in vivo. This revealed that import of Cho into the chloroplast is a major constraint on GlyBet synthesis, the import rate being approximately 100-fold lower than the rates of Cho phosphorylation and transport into the vacuole, with which import competes. Simulation studies suggested that, were the chloroplast transport limitation corrected, additional engineering interventions would still be needed to achieve levels of GlyBet as high as those in plants that accumulate GlyBet naturally. This study reveals the rigidity of the Cho metabolism network and illustrates how computer modeling can help guide rational metabolic engineering design.

Betaine↗

Re-engineering healthcare pipelnes: why trajectory selection is as important as process selection in enabling effective transfer of best practice.

PURPOSE: To demonstrate that effective re-engineering of healthcare pipelines requires selecting both the "best" process (how we shall do it in the future) and the best trajectory for change (how we get from here to there). DESIGN/METHODOLOGY/APPROACH: Exploitation of Braess' Paradox to identify strategic factors necessary to enable change in the re-engineering of NHS healthcare pipelines. FINDINGS: Route to maximising the chance of achieving effective change is displayed via a Johari Window. Each cell is then related to no change (despite significant investment); failure; or successful implementation. RESEARCH LIMITATIONS/IMPLICATIONS: The demonstrator pipeline is an NHS cataract repair supply chain. However, via the "Power of Analogy" concept the NHS scenario is readily related to a substantial number of industrial case studies. PRACTICAL IMPLICATIONS: The need to understand both process and trajectory is the key to effective re-engineering of pipelines. All "actors" in re-engineering programmes should acquire this knowledge and benefit from the new way of doing things. ORIGINALITY/VALUE: Formalises the strategic route to enabling healthcare delivery "best practice".

Health Care Reform↗

An engineered sorbitol cycle alters sugar composition, not growth, in transformed tobacco.

Many efforts have been made to engineer stress tolerance by accumulating polyols. Transformants that accumulate polyols often show growth inhibition, because polyols are synthesized as a dead-end product in plants that do not naturally accumulate polyols. Here, we show a novel strategy in which a sorbitol cycle was engineered by introducing apple cDNA encoding NAD-dependent sorbitol dehydrogenase (SDH) in addition to sorbitol-6-phosphate dehydrogenase (S6PDH). Tobacco plants transformed only with S6PDH showed growth inhibition, and very few transformants were obtained. In contrast, many transgenic plants with both S6PDH and SDH were easily obtained, and their growth was normal despite their accumulation of sorbitol. Interestingly, the engineered sorbitol cycle enhanced the accumulation of sucrose instead of fructose that was expected to be increased. Sucrose, rather than fructose, was also increased in the immature fruit of tomato plants transformed with an antisense fructokinase gene in which the phosphorylation of fructose was inhibited. A common phenomenon was observed in the metabolic engineering of two different pathways, showing the presence of homeostatic regulation of fructose levels.

Antisense Elements (Genetics)↗

Ecosystem engineers as selective agents: the effects of leaf litter on emergence time and early growth in Impatiens capensis.

By physically modifying the abiotic environment, ecosystem engineers can have dramatic effects on the distribution and abundance of species in a community. However, ecosystem engineering can also change the selective environment and evolutionary dynamics of affected species, although this remains relatively understudied. Here, we examine the potential for an ecosystem engineer - oak trees - to affect the evolutionary dynamics of the herbaceous, understory annual, Impatiens capensis, through leaf litter deposition. Using a quantitative genetic experimental approach, we found that: (i) the presence of leaf litter significantly affected a suite of germination, growth and phenological traits in I. capensis; (ii) I. capensis does not exhibit performance trade-offs across litter and bare soil environments in the form of negative across-environment genetic correlations; (iii) the presence or absence of leaf litter significantly alters the pattern of natural selection germination timing and hypocotyl length; and (iv) the frequency of leaf litter environments can dramatically change which combinations of hypocotyl length lead to highest mean fitness across both bare soil and leaf litter environments. More generally, our results demonstrate the potential for ecosystem engineers to alter both the ecological and the evolutionary dynamics of the species they affect.

Ecosystem↗

A study of medical and health queries to web search engines.

This paper reports findings from an analysis of medical or health queries to different web search engines. We report results: (i). comparing samples of 10000 web queries taken randomly from 1.2 million query logs from the AlltheWeb.com and Excite.com commercial web search engines in 2001 for medical or health queries, (ii). comparing the 2001 findings from Excite and AlltheWeb.com users with results from a previous analysis of medical and health related queries from the Excite Web search engine for 1997 and 1999, and (iii). medical or health advice-seeking queries beginning with the word 'should'. Findings suggest: (i). a small percentage of web queries are medical or health related, (ii). the top five categories of medical or health queries were: general health, weight issues, reproductive health and puberty, pregnancy/obstetrics, and human relationships, and (iii). over time, the medical and health queries may have declined as a proportion of all web queries, as the use of specialized medical/health websites and e-commerce-related queries has increased. Findings provide insights into medical and health-related web querying and suggests some implications for the use of the general web search engines when seeking medical/health information.

Europe↗