Organic tissues in rotating bioreactors: fluid-mechanical aspects, dynamic growth models, and morphological evolution

This analysis deals with advances in tissue-engineering models and computational methods as well as with novel results on the relative importance of "controlling forces" in the growth of organic constructs. Specifically, attention is focused on the rotary culture system, because this technique has proven to be the most practical solution for providing a suitable culture environment supporting three-dimensional tissue assemblies. From a numerical point of view, the growing biological specimen gives rise to a moving boundary problem. A "volume-of-fraction" method is specifically and carefully developed according to the complex properties and mechanisms of organic tissue growth and, in particular, taking into account the sensitivity of the construct/liquid interface to the effect of the fluid-dynamic shear stress (it induces changes in tissue metabolism and function that elicit a physiological response from the biological cells). The present study uses available data to introduce a set of growth models. The surface conditions are coupled to the transfer of mass and momentum at the specimen/culture-medium interface and lead to the introduction of a group of differential equations for the nutrient concentration around the sample and for the evolution of tissue mass displacement. The models are then used to show how the proposed surface kinetic laws can predict (through sophisticated numerical simulations) many of the known characteristics of biological tissues grown using rotating-wall perfused vessel bioreactors. This procedure provides a validation of the models and associated numerical method and also gives insight into the mechanisms of the phenomena. The interplay between the increasing size of the tissue and the structure of the convective field is investigated. It is shown that this interaction is essential in determining the time evolution of the tissue shape. The size of the growing specimen plays a critical role with regard to the intensity of convection and the related shear stresses. Convective effects, in turn, are found to impact growth rates, tissue size, and morphology, as well as the mechanisms driving growth. The method exhibits novel capabilities to predict and elucidate experimental observations and to identify cause-and-effect relationships.     

Interferometric Studies of Growth Kinetics and Surface Morphology in Macromolecular Crystal Growth: Canavalin, Thaumatin, and Turnip Yellow Mosaic Virus

In situ laser Michelson interferometry was utilized to investigate mechanisms of growth and surface morphology in protein and virus crystallization, These included plant proteins canavalin and thaumatin and turnip yellow mosaic virus. The experimental apparatus allowed us to obtain interferometric patterns and investigate growth kinetics from growing macromolecular crystals as small as 20 μm. For the crystallization of canavalin, dislocations are the sources of growth steps on the surfaces. Supersaturation and time dependencies of the normal growth rates, tangential growth step velocities, and the slopes of the dislocation hillocks were measured. The kinetic coefficient β (rate of incorporation of protein molecules into the growing crystal) was estimated for canavalin to be 9 × 10^-4 cm/sec. This is among the first estimates of such fundamental kinetic parameters for macromolecular crystallization. The change in the activities of dislocation sources under different growth conditions was also analyzed. Michelson interferometry was clearly demonstrated to be a useful tool for quantitative studies of macromolecular crystal growth.     

智能多肽的自组装机理及其生物学应用

智能多肽是指智能响应外界刺激并做出相应回应的多肽。由于其形成过程为自发的自组装,故智能多肽又可称为自组装多肽。智能多肽的氨基酸构成使其拥有良好的生物相容性及生物可降解性,作为构筑基元拼接成为功能性材料,在新型生物材料方面展示出了广阔的应用前景。概括了智能多肽的性质、自组装机理及应用,重点阐述了它在生物能源、生物医学工程和分离工程上的应用,以期在系统认识智能多肽的基础上,发掘其应用潜能,突破开发瓶颈。     

Modeling the Effect of Curvature on the Collective Behavior of Cells Growing New Tissue

The growth of several biological tissues is known to be controlled in part by local geometrical features, such as the curvature of the tissue interface. This control leads to changes in tissue shape that in turn can affect the tissue’s evolution. Understanding the cellular basis of this control is highly significant for bioscaffold tissue engineering, the evolution of bone microarchitecture, wound healing, and tumor growth. Although previous models have proposed geometrical relationships between tissue growth and curvature, the role of cell density and cell vigor remains poorly understood. We propose a cell-based mathematical model of tissue growth to investigate the systematic influence of curvature on the collective crowding or spreading of tissue-synthesizing cells induced by changes in local tissue surface area during the motion of the interface. Depending on the strength of diffusive damping, the model exhibits complex growth patterns such as undulating motion, efficient smoothing of irregularities, and the generation of cusps. We compare this model with in vitro experiments of tissue deposition in bioscaffolds of different geometries. By including the depletion of active cells, the model is able to capture both smoothing of initial substrate geometry and tissue deposition slowdown as observed experimentally.     

Distinguishing crystallographic from biological interfaces in protein complexes: role of intermolecular contacts and energetics for classification

Background

Study of macromolecular assemblies is fundamental to understand functions in cells. X-ray crystallography is the most common technique to solve their 3D structure at atomic resolution. In a crystal, however, both biologically-relevant interfaces and non-specific interfaces resulting from crystallographic packing are observed. Due to the complexity of the biological assemblies currently tackled, classifying those interfaces, i.e. distinguishing biological from crystal lattice interfaces, is not trivial and often prone to errors. In this context, analyzing the physico-chemical characteristics of biological/crystal interfaces can help researchers identify possible features that distinguish them and gain a better understanding of the systems.

Results

In this work, we are providing new insights into the differences between biological and crystallographic complexes by focusing on “pair-properties” of interfaces that have not yet been fully investigated. We investigated properties such intermolecular residue-residue contacts (already successfully applied to the prediction of binding affinities) and interaction energies (electrostatic, Van der Waals and desolvation). By using the XtalMany and BioMany interface datasets, we show that interfacial residue contacts, classified as a function of their physico-chemical properties, can distinguish between biological and crystallographic interfaces. The energetic terms show, on average, higher values for crystal interfaces, reflecting a less stable interface due to crystal packing compared to biological interfaces. By using a variety of machine learning approaches, we trained a new interface classification predictor based on contacts and interaction energetic features. Our predictor reaches an accuracy in classifying biological vs crystal interfaces of 0.92, compared to 0.88 for EPPIC (one of the main state-of-the-art classifiers reporting same performance as PISA).

Conclusion

In this work we have gained insights into the nature of intermolecular contacts and energetics terms distinguishing biological from crystallographic interfaces. Our findings might have a broader applicability in structural biology, for example for the identification of near native poses in docking. We implemented our classification approach into an easy-to-use and fast software, freely available to the scientific community from http://github.com/haddocking/interface-classifier.

     

Fragmentation-tree density representation for crystallographic modelling of bound ligands

The identification and modelling of ligands into macromolecular models is important for understanding molecule's function and for designing inhibitors to modulate its activities. We describe new algorithms for the automated building of ligands into electron density maps in crystal structure determination. Location of the ligand-binding site is achieved by matching numerical shape features describing the ligand to those of density clusters using a "fragmentation-tree" density representation. The ligand molecule is built using two distinct algorithms exploiting free atoms with inter-atomic connectivity and Metropolis-based optimisation of the conformational state of the ligand, producing an ensemble of structures from which the final model is derived. The method was validated on several thousand entries from the Protein Data Bank. In the majority of cases, the ligand-binding site could be correctly located and the ligand model built with a coordinate accuracy of better than 1 ?. We anticipate that the method will be of routine use to anyone modelling ligands, lead compounds or even compound fragments as part of protein functional analyses or drug design efforts.     

工程化骨软骨生理微环境构建方法及研究进展

骨组织工程是通过在体外构建有正常组织功能或疾病生理特点的临床模型,用以药物筛选,或研究疾病发生发展过程。骨骼肌肉系统是载重系统,其功能与组织结构、细胞外基质等密切相关。在构建骨组织体外模型时,需要结合骨、软骨及其他构成成分的生理微环境,表现关节骨软骨接合处的生理特点及作用机制,进而模拟正常及病理状态下骨组织系统对刺激的反应。本综述从骨软骨组织的生理构造入手,阐述了骨软骨连接处在退行性关节病变发生发展过程中的作用,并系统的论述了体外构建三维骨软骨组织的方法及这些方法的优势和局限性,为体外构建骨软骨组织工程在临床上应用提供支持。     

Computational modeling of chemical reactions and interstitial growth and remodeling involving charged solutes and solid-bound molecules

Mechanobiological processes are rooted in mechanics and chemistry, and such processes may be modeled in a framework that couples their governing equations starting from fundamental principles. In many biological applications, the reactants and products of chemical reactions may be electrically charged, and these charge effects may produce driving forces and constraints that significantly influence outcomes. In this study, a novel formulation and computational implementation are presented for modeling chemical reactions in biological tissues that involve charged solutes and solid-bound molecules within a deformable porous hydrated solid matrix, coupling mechanics with chemistry while accounting for electric charges. The deposition or removal of solid-bound molecules contributes to the growth and remodeling of the solid matrix; in particular, volumetric growth may be driven by Donnan osmotic swelling, resulting from charged molecular species fixed to the solid matrix. This formulation incorporates the state of strain as a state variable in the production rate of chemical reactions, explicitly tying chemistry with mechanics for the purpose of modeling mechanobiology. To achieve these objectives, this treatment identifies the specific theoretical and computational challenges faced in modeling complex systems of interacting neutral and charged constituents while accommodating any number of simultaneous reactions where reactants and products may be modeled explicitly or implicitly. Several finite element verification problems are shown to agree with closed-form analytical solutions. An illustrative tissue engineering analysis demonstrates tissue growth and swelling resulting from the deposition of chondroitin sulfate, a charged solid-bound molecular species. This implementation is released in the open-source program FEBio (www.febio.org). The availability of this framework may be particularly beneficial to optimizing tissue engineering culture systems by examining the influence of nutrient availability on the evolution of inhomogeneous tissue composition and mechanical properties, the evolution of construct dimensions with growth, the influence of solute and solid matrix electric charge on the transport of cytokines, the influence of binding kinetics on transport, the influence of loading on binding kinetics, and the differential growth response to dynamically loaded versus free-swelling culture conditions.     

Incorporation of microcrystals by growing protein and virus crystals

In the course of time-lapse video and atomic force microscopy (AFM) investigations of macromolecular crystal growth, we frequently observed the sedimentation of microcrystals and three-dimensional nuclei onto the surfaces of much larger, growing protein or virus crystals. This was followed by the direct incorporation over time of the smaller crystals into the bulk of the larger crystals. In some cases, clear indications were present that upon absorption of the small crystal onto the surface of the larger, there was proper alignment of the respective lattices, and consolidation proceeded without observable defect formation, i.e., the two lattices knitted together without discontinuity. In the case of at least one virus crystal, cubic satellite tobacco mosaic virus (STMV), addition of three-dimensional nuclei and subsequent expansion provided the principal growth mechanism at high supersaturation. This process has not been reported for growth from solution of conventional crystals. In numerous other instances, the lattices of the small and larger crystals were obviously misaligned, and incorporation occurred with the formation of some defect. This phenomenon of small crystals physically embedded in larger crystals could only degrade the overall diffraction and materials properties of macromolecular crystals.     

A study of the interface strength between protein and mineral in biological materials

Bone, tooth, mineralized tendon and sea shells are nanocomposites of protein and mineral with superior mechanical properties. As the mineral is so small at nanoscale, the volume fraction of the protein-mineral interface in the bulk materials can be enormously large; therefore, the mechanics of the interface should be critically important for the integrity of these biomaterials. Currently, people do not have a good understanding of the interface between protein and mineral, a hybrid interface between organic and inorganic constituents in biological materials. In this paper, a tension-shear chain (TSC) model is introduced into the Dugdale model for estimating the fracture energy of biomaterials. The strength of the hybrid interface is then studied with a "soft-hard" bi-layer fracture model, by which we find for the first time that the interface strength depends on both the size and geometry of the mineral crystal, and has been highly optimized through the miniaturization of mineral at nanoscale. This study may provide important insights into the mechanics of bone and tooth at small scale for tissue engineering in biomedical applications.     

Computer simulation studies of self-assembling macromolecules

Coarse-grained (CG) molecular models are now widely used to understand the structure and functionality of macromolecular self-assembling systems. In the last few years, significant efforts have been devoted to construct quantitative CG models based on data from molecular dynamics (MD) simulations with more detailed all-atom (AA) intermolecular force fields as well as experimental thermodynamic data. We review some of the recent progress pertaining to the MD simulation of self-assembling macromolecular systems, using as illustrations the application of CG models to probe surfactant and lipid self-assembly including liposome and dendrimersome formation as well as the interaction of biomembranes with nanoparticles.     

电磁场曝露对生物组织电磁特性的影响

电磁辐射严重影响着人体的健康.电磁场生物效应的发生机制与电磁场本身的特性相关,同时也与生物组织在电磁场作用下电磁特性的改变密切相关.生物体内的信号分子、自由基以及磁颗粒等处于外加电磁场中时其电磁特性会发生变化,尤其是不同频率电磁场曝露作用下生物组织的导电、介电以及磁学等特性会有非常显著的区别.明确不同频率电磁场作用下生物组织电磁特性的变化规律是研究电磁场生物效应发生机制以及预防问题的关键.综述了近年来电磁场对于生物组织电磁特性影响的研究成果,并对未来的研究方向做了展望.     

Evolution of bioengineering at UCSD: opening new vistas

Before this, the field of bioengineering refers to biomedical engineering of prosthetic devices in physiology. In addition to exciting applications of engineering principles, UCSD Department of Bioengineering began to extend the notion of engineering models of physiological systems to physiological processes. This led to a conceptual shift in the discipline and contributed to the areas of tissue and physiological process engineering. In 1988, Dr. Shu Chien and Richard Skalak joined UCSD to begin research and education on cellular and molecular bioengineering, especially, mechanobiology. Dr. Fung and Dr. Skalak initiated the new field of tissue engineering. These two decades of evolution of bioengineering and its growth across the country was spearheaded by the Whitaker Foundation, whose leitmotif was the building of bioand biomedical engineering across the country. We have garnered other accomplishments in the following fields: regenerative medicine; bioinspired artificial extracellular matrices; flexible bioelectronics and tatoos; cells show how to synchronize biological clocks; and systems medicine.     

Repair of impurity-poisoned protein crystal surfaces

The surface morphology of Bence-Jones protein (BJP) crystals was investigated during growth and dissolution by using in situ atomic force microscopy (AFM). It was shown that over a wide supersaturation range, impurities adsorb on the crystalline surface and ultimately form an impurity adsorption layer that prevents further growth of the crystal. At low undersaturations, this impurity adsorption layer prevents dissolution. At greater undersaturation, dissolution takes place around large particles incorporated into the crystal, leading to etch pits with impurity-free bottoms. On restoration of supersaturation conditions, two-dimensional nucleation takes place on the impurity-free bottoms of these etch pits. After new growth layers fill in the etch pits, they cover the impurity-poisoned top layer of the crystal face. This leads to the resumption of its growth. Formation of an impurity-adsorption layer can explain the termination of growth of macromolecular crystals that has been widely noted. Growth-dissolution-growth cycles could be used to produce larger crystals that otherwise would have stopped growing because of impurity poisoning.     

A simple apparatus for controlling nucleation and size in protein crystal growth

A simple device is described for controlling vapor equilibrium in macromolecular crystallization as applied to the protein crystal growth technique commonly referred to as the "hanging drop" method. Crystal growth experiments with hen egg white lysozyme have demonstrated control of the nucleation rate. Nucleation rate and final crystal size have been found to be highly dependent upon the rate at which critical supersaturation is approached. Slower approaches show a marked decrease in the nucleation rate and an increase in crystal size.     

Effect of biofilm growth on steady-state biofilm models

The results of numerical simulations for a growing biological film are presented to justify the use of steady-state biofilm models for approximating the behavior of both unlimited and shear-limited biofilms. For an unlimited biofilm we show that although the total biofilm thickness may continue to increase over time, the active biofilm volume will reach a constant value. We also show that the profile of active microorganisms within the biofilm will become constant with respect to the biofilm/fluid interface and simply move outward as the biofilm thickness increases. For a shear-limited biofilm we similarly show that once a "limiting" thickness has been reached the active biofilm volume, substrate consumption, and profile of active microorganisms within the biofilm will also be independent of the biofilm thickness.     

Silk: molecular organization and control of assembly

The interface between the science and engineering of biology and materials is an area of growing interest. One of the goals of this field is to utilize biological synthesis and processing of polymers as a route to gain insight into topics such as molecular recognition, self-assembly and the formation of materials with well-defined architectures. The biological processes involved in polymer synthesis and assembly can offer important information on fundamental interactions involved in the formation of complex material architectures, as well as practical knowledge into new and important materials related to biomaterial uses and tissue engineering needs. Classic approaches in biology, including genetic engineering, controlled microbial physiology and enzymatic synthesis, are prototypical methods used to control polymer structure and chemistry, including stereoselectivity and regioselectivity, to degrees unattainable using traditional synthetic chemistry. This type of control can lead to detailed and systematic studies of the formation of the structural hierarchy in materials and the subsequent biological responses to these materials.     

Molecular-level thermodynamic and kinetic parameters for the self-assembly of apoferritin molecules into crystals

The self-assembly of apoferritin molecules into crystals is a suitable model for protein crystallization and aggregation; these processes underlie several biological and biomedical phenomena, as well as for protein and virus self-assembly. We use the atomic force microscope in situ, during the crystallization of apoferritin to visualize and quantify at the molecular level the processes responsible for crystal growth. To evaluate the governing thermodynamic parameters, we image the configuration of the incorporation sites, "kinks", on the surface of a growing crystal. We show that the kinks are due to thermal fluctuations of the molecules at the crystal-solution interface. This allows evaluation of the free energy of the intermolecular bond phi=3.0 k(B)T=7.3 kJ/mol. The crystallization free energy, extracted from the protein solubility, is -42 kJ/mol. Published determinations of the second virial coefficient and the protein solubility between 0 and 40 degrees C revealed that the enthalpy of crystallization is close to zero. Analyses based on these three values suggest that the main component in the crystallization driving force is the entropy gain of the water molecules bound to the protein molecules in solution and released upon crystallization. Furthermore, monitoring the incorporation of individual molecules in to the kinks, we determine the characteristic frequency of attachment of individual molecules at one set of conditions. This allows a correlation between the mesoscopic kinetic coefficient for growth and the molecular-level thermodynamic and kinetic parameters determined here. We found that step growth velocity, scaled by the molecular size, equals the product of the kink density and attachment frequency, i.e. the latter pair are the molecular-level parameters for self-assembly of the molecules into crystals.     

Effects of boundary conditions on the estimation of the planar biaxial mechanical properties of soft tissues

Evaluation and simulation of the multiaxial mechanical behavior of native and engineered soft tissues is becoming more prevalent. In spite of this growing use, testing methods have not been standardized and methodologies vary widely. The strong influence of boundary conditions were recently underscored by Waldman et al. [2002, J. Materials Science: Materials in Medicine 13, pp. 933-938] wherein substantially different experimental results were obtained using different sample gripping methods on the same specimens. As it is not possible to experimentally evaluate the effects of different biaxial test boundary conditions on specimen internal stress distributions, we conducted numerical simulations to explore these effects. A nonlinear Fung-elastic constitutive model (Sun et al., 2003, JBME 125, pp. 372-380, which fully incorporated the effects of in-plane shear, was used to simulate soft tissue mechanical behavior. Effects of boundary conditions, including varying the number of suture attachments, different gripping methods, specimen shapes, and material axes orientations were examined. Results demonstrated strong boundary effects with the clamped methods, while suture attachment methods demonstrated minimal boundary effects. Suture-based methods appeared to be best suited for biaxial mechanical tests of biological materials. Moreover, the simulations demonstrated that Saint-Venant's effects depended significantly on the material axes orientation. While not exhaustive, these comprehensive simulations provide experimentalists with additional insight into the stress-strain fields associated with different biaxial testing boundary conditions, and may be used as a rational basis for the design of biaxial testing experiments.