丝纤维支架材料的制备技术及其应用研究进展

丝纤维特别是丝素蛋白和蜘蛛丝蛋白作为具有良好生物相容性的高分了生物材料在组织工程和生物医学领域里有着广泛的应用。本文阐述了近年来在组织工程研究中所涉及的利用丝纤维进行支架材料制备、细胞培养和体内植入检测手段等方面的研究概况。     

Enhancement of cell growth in tissue-engineering constructs under direct perfusion: Modeling and simulation

Perfusion bioreactors improve mass transfer in cell-scaffold constructs. We developed a mathematical model to simulate nutrient flow through cellular constructs. Interactions among cell proliferation, nutrient consumption, and culture medium circulation were investigated. The model incorporated modified Contois cell-growth kinetics that includes effects of nutrient saturation and limited cell growth. Nutrient uptake was depicted through the Michaelis-Menton kinetics. To describe the culture medium convection, the fluid flow outside the cell-scaffold construct was described by the Navier-Stokes equations, while the fluid dynamics within the construct was modeled by Brinkman's equation for porous media flow. Effects of the media perfusion were examined by including time-dependant porosity and permeability changes due to cell growth. The overall cell volume was considered to consist of cells and extracellular matrices (ECM) as a whole without treating ECM separately. Numerical simulations show when cells were cultured subjected to direct perfusion, they penetrated to a greater extent into the scaffold and resulted in a more uniform spatial distribution. The cell amount was increased by perfusion and ultimately approached an asymptotic value as the perfusion rates increased in terms of the dimensionless Peclet number that accounts for the ratio of nutrient perfusion to diffusion. In addition to enhancing the nutrient delivery, perfusion simultaneously imposes flow-mediated shear stress to the engineered cells. Shear stresses were found to increase with cell growth as the scaffold void space was occupied by the cell and ECM volumes. The macro average stresses increased from 0.2 mPa to 1 mPa at a perfusion rate of 20 microm/s with the overall cell volume fraction growing from 0.4 to 0.7, which made the overall permeability value decrease from 1.35 x 10(-2)cm(2) to 5.51 x 10(-4)cm(2). Relating the simulation results with perfusion experiments in literature, the average shear stresses were below the critical value that would induce the chondrocyte necrosis.     

Biofunctionalized nanoneedles for the direct and site-selective delivery of probes into living cells

Background

Accessing the interior of live cells with minimal intrusiveness for visualizing, probing, and interrogating biological processes has been the ultimate goal of much of the biological experimental development.

Scope of review

The recent development and use of the biofunctionalized nanoneedles for local and spatially controlled intracellular delivery brings in exciting new opportunities in accessing the interior of living cells. Here we review the technical aspect of this relatively new intracellular delivery method and the related demonstrations and studies and provide our perspectives on the potential wide applications of this new nanotechnology-based tool in the biological field, especially on its use for high-resolution studies of biological processes in living cells.

Major conclusions

Different from the traditional micropipette-based needles for intracellular injection, a nanoneedle deploys a sub-100-nm-diameter solid nanowire as a needle to penetrate a cell membrane and to transfer and deliver the biological cargo conjugated onto its surface to the target regions inside a cell. Although the traditional micropipette-based needles can be more efficient in delivery biological cargoes, a nanoneedle-based delivery system offers an efficient introduction of biomolecules into living cells with high spatiotemporal resolution but minimal intrusion and damage. It offers a potential solution to quantitatively address biological processes at the nanoscale.

General significance

The nanoneedle-based cell delivery system provides new possibilities for efficient, specific, and precise introduction of biomolecules into living cells for high-resolution studies of biological processes, and it has potential application in addressing broad biological questions.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.     

光学显微镜技术在活细胞单分子检测中的应用

单分子检测是一门以高度的时间以及空间分辨率研究生物单分子的技术。近来,科学技术的探索发展使我们可以观察、检测甚至操纵单个分子并且研究它们的构象变化和动力学行为。这一发展使得以前被传统系综研究体系平均化所隐藏的新信息被揭示出来。单分子检测技术的发展已经揭开了生命科学研究的新篇章。在本文中,我们将介绍有关活细胞中单分子检测技术的发展以及活细胞内单分子检测的现状。     

Dynamics of human DNA topoisomerases IIalpha and IIbeta in living cells

DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (alpha and beta) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIalpha and IIbeta behaved similarly in interphase but differently in mitosis, where only topo IIalpha was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIalpha was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.     

Marrow-derived stem cell motility in 3D synthetic scaffold is governed by geometry along with adhesivity and stiffness

Design of 3D scaffolds that can facilitate proper survival, proliferation, and differentiation of progenitor cells is a challenge for clinical applications involving large connective tissue defects. Cell migration within such scaffolds is a critical process governing tissue integration. Here, we examine effects of scaffold pore diameter, in concert with matrix stiffness and adhesivity, as independently tunable parameters that govern marrow‐derived stem cell motility. We adopted an “inverse opal” processing technique to create synthetic scaffolds by crosslinking poly(ethylene glycol) at different densities (controlling matrix elastic moduli or stiffness) and small doses of a heterobifunctional monomer (controlling matrix adhesivity) around templating beads of different radii. As pore diameter was varied from 7 to 17 µm (i.e., from significantly smaller than the spherical cell diameter to approximately cell diameter), it displayed a profound effect on migration of these stem cells—including the degree to which motility was sensitive to changes in matrix stiffness and adhesivity. Surprisingly, the highest probability for substantive cell movement through pores was observed for an intermediate pore diameter, rather than the largest pore diameter, which exceeded cell diameter. The relationships between migration speed, displacement, and total path length were found to depend strongly on pore diameter. We attribute this dependence to convolution of pore diameter and void chamber diameter, yielding different geometric environments experienced by the cells within. Bioeng. 2011; 108:1181–1193. © 2010 Wiley Periodicals, Inc.     

The effect of substrate and epidermal growth factor on human placental trophoblast cells in culture

Summary Attempts were made to select for trophoblast cells in cultures of mixed cell populations derived from preterm (7 to 12 wk) or term human placentas. Epidermal growth factor added to cultures on solid or porous supports caused proliferation of epithelial-type cells to give a confluent monolayer but did not increase the expression of differentiated function. The presence or absence of placental basement membrane collagen as substrate made little apparent difference; however a porous basement membrane collagen support led to increased differentiated function. Initial production of human chorionic gonadotrophin was increased and after 4 wk in culture a substantial proportion of the cells exhibited alkaline phosphatase activity. Epidermal growth factor and a substrate of placental basement membrane collagen on a porous support favorably influence the growth and differentiation of human trophoblast cells in culture. This work was supported by funds from the Medical Research Council of New Zealand which also provided support for Dr. Truman as a Postdoctoral Fellow.     

新型旋转壁式生物反应器内三维组织工程骨的构建

利用微载体悬浮培养法将成骨细胞在旋转壁式生物反应器内进行大规模扩增,并检测细胞的组织形态和生物功能.然后以此作为种子细胞,分别以2×10⁶个/ml和1×10⁶个/ml两种密度接种到支架材料上,于旋转壁式生物反应器(RWV)内进行三维组织工程骨的构建.并将所构建的骨组织分别进行倒置显微镜(inverted microscope)、扫描电镜(SEM)、碱性磷酸酶(ALP)、矿化结构和AO/EB双重荧光染色等生物学性能检测,以及对培养过程的营养物质代谢情况进行监控和分析.结果表明,在RWV中培养的骨组织生长良好,分泌大量胶原纤维,并有矿化基质和新骨样组织形成. 由上述结果可断定,通过RWV内部流体对流所产生的应力刺激,可提高成骨细胞碱性磷酸酶的活性表达,并加速矿化结节的形成,从而完成成骨细胞的快速增殖与分化以及工程化组织的三维构建.     

Preclinical three-dimensional colorectal cancer model: The next generation of in vitro drug efficacy evaluation

Colorectal cancer (CRC), the third most common cancer diagnosed in both men and women in the United States, shows a highly ineffective therapeutic management. In these years neither substantial improvements nor new therapeutic approaches have been provided to patients. Performing the early lead discovery phases of new cancer drugs in cellular models, resembling as far as possible the real in vivo tumor environment, may be more effective in predicting their future success in the later clinical phases. In this review, we critically describe the most representative bioengineered models for anticancer drug screening in CRC from the conventional two-dimensional models to the new-generation three-dimensional scaffold-based ones. The scaffold aims to replace the extracellular matrix, thus influencing the biomechanical, biochemical, and biological properties of cells and tissues. In this scenario, we believe that reconstitution of tumor condition is mandatory for an alternative in vitro methods to study cancer development and therapeutic strategies.     

Excitability and isometric contractile properties of mammalian skeletal muscle constructs engineered in vitro

Summary Our purpose was to engineer three-dimensional skeletal muscle tissue constructs from primary cultures of adult rat myogenic precursor cells, and to measure their excitability and isometric contractile properties. The constructs, termed myooids, were muscle-like in appearance, excitability, and contractile function. The myooids were 12 mm long and ranged in diameter from 0.1 to 1 mm. The myooids were engineered with synthetic tendons at each end to permit the measurement of isometric contractile properties. Within each myooid the myotubes and fibroblasts were supported by an extracellular matrix generated by the cells themselves, and did not require a preexisting scaffold to define the size, shape, and general mechanical properties of the resulting structure. Once formed, the myooids contracted spontaneously at approximately 1 Hz, with peak-to-peak force amplitudes ranging from 3 to 30 μN. When stimulated electrically the myooids contracted to produce force. The myooids (n=14) had the following mean values: diameter of 0.49 mm, rheobase of 1.0 V/mm, chronaxie of 0.45 ms, twitch force of 215 μN, maximum isometric force of 440 μN, resting baseline force of 181 μN, and specific force of 2.9kN/m². The mean specific force was approximately 1% of the specific force generated by control adult rat muscle. Based on the functional data, the myotubes in the myooids appear to remain arrested in an early developmental state due to the absence of signals to promote expression of adult myosin isoforms.     

Biomaterials for the programming of cell growth in oral tissues: The possible role of APA

Examples of programmed tissue response after the interaction of cells with biomaterials are a hot topic in current dental research. We propose here the use of anodic porous alumina (APA) for the programming of cell growth in oral tissues. In particular, APA may trigger cell growth by the controlled release of specific growth factors and/or ions. Moreover, APA may be used as a scaffold to promote generation of new tissue, due to the high interconnectivity of pores and the high surface roughness displayed by this material.     

The biological standard of living in Taiwan under Japanese occupation

This paper presents evidence on the biological standard of living in Taiwan from 1842 to 1931 using Taiwanese height and weight data collected by the Japanese authorities from 1921 to 1931. This study shows that in the late Ch'ing adult heights were not increasing over time, while the adult heights of those born after the Japanese takeover did begin to increase rapidly. Evidence from children's heights confirms that this growth in height continued through the 1920s. The body mass index of Taiwanese, however, did not increase in the 1920s. By most measures, the biological standard of living was better in the north of the island. Comparison with modern data shows that heights have continued to increase.     

Direct noninvasive measurement and numerical modeling of depth-dependent strains in layered agarose constructs

Biomechanical factors play an important role in the growth, regulation, and maintenance of engineered biomaterials and tissues. While physical factors (e.g. applied mechanical strain) can accelerate regeneration, and knowledge of tissue properties often guide the design of custom materials with tailored functionality, the distribution of mechanical quantities (e.g. strain) throughout native and repair tissues is largely unknown. Here, we directly quantify distributions of strain using noninvasive magnetic resonance imaging (MRI) throughout layered agarose constructs, a model system for articular cartilage regeneration. Bulk mechanical testing, giving both instantaneous and equilibrium moduli, was incapable of differentiating between the layered constructs with defined amounts of 2% and 4% agarose. In contrast, MRI revealed complex distributions of strain, with strain transfer to softer (2%) agarose regions, resulting in amplified magnitudes. Comparative studies using finite element simulations and mixture (biphasic) theory confirmed strain distributions in the layered agarose. The results indicate that strain transfer to soft regions is possible in vivo as the biomaterial and tissue changes during regeneration and maturity. It is also possible to modulate locally the strain field that is applied to construct-embedded cells (e.g. chondrocytes) using stratified agarose constructs.     

Evaluation of cellular adhesion and organization in different microporous polymeric scaffolds

The lack of prediction accuracy during drug development and screening risks complications during human trials, such as drug‐induced liver injury (DILI), and has led to a demand for robust, human cell‐based, in vitro assays for drug discovery. Microporous polymer‐based scaffolds offer an alternative to the gold standard flat tissue culture plastic (2D TCPS) and other 3D cell culture platforms as the porous material entraps cells, making it advantageous for automated liquid handlers and high‐throughput screening (HTS). In this study, we optimized the surface treatment, pore size, and choice of scaffold material with respect to cellular adhesion, tissue organization, and expression of complex physiologically relevant (CPR) outcomes such as the presence of bile canaliculi‐like structures. Poly‐l‐ lysine and fibronectin (FN) coatings have been shown to encourage cell attachment to the underlying substrate. Treatment of the scaffold surface with NaOH followed with a coating of FN improved cell attachment and penetration into pores. Of the two pore sizes we investigated (A: 104 ± 4 μm; B: 175 ± 6 μm), the larger pore size better promoted cell penetration while limiting tissue growth from reaching the hypoxia threshold. Finally, polystyrene (PS) proved to be conducive to cell growth, penetration into the scaffold, and yielded CPR outcomes while being a cost‐effective choice for HTS applications. These observations provide a foundation for optimizing microporous polymer‐based scaffolds suitable for drug discovery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:505–514, 2018     

A novel artificial substrate for cell culture: Effects of substrate flexibility/malleability on cell growth and morphology

Summary Gels of glyoxyl agarose (GA) are evaluated as a novel flexible substrate for cell culture with physical properties comparable to extracellular matrix (ECM) gels. We show here that cells adhere well to pure GA gels; in addition, specific interactions involving matrix receptors can be studied when individual matrix molecules are bound to the gel covalently. When cells are grown on such substrates, morphology is comparable to that observed on “natural” matrix gels (reconstituted gels of collagen type I or of Matrigel): rather than being flattened as in monolayer cultures on tissue culture plastic the cells assume a rounded morphology and tend to form tissue-like aggregates. The effects of the artificial matrix gels are discussed in the context of previous publications on cell interactions with the extracellular matrix, suggesting that in addition to specific recognition of matrix molecules the physical properties of ECM by themselves can be decisive for cell differentiation. We conclude that gels of glycoxyl agarose a) provide a useful model to mimic the physical properties of matrix gels without the presence of specific adhesion factors; b) may be useful as a general, non-specific ECM allowing cells to be cultured in vitro under conditions favorable for differentiation; and c) allow to design a variety of “synthetic” ECM models composed of a chemically defined gel matrix, which can be supplemented with covalently bound molecules to be recognized by cell surface receptors.     

生计方式对农户生活能源消费模式的影响——以甘南高原为例

生计方式变迁对农户的生活能源消费模式产生深远影响,从而给农村能源与环境问题带来新的挑战。以地处青藏高原东缘的甘南高原为研究区,基于农户调查资料,采用STIRPAT模型与二元logistic模型分析了生计方式对农户的生活能源消费量及生活能源消费模式选择的影响。结果发现:(1)随着非农化水平的提高,甘南高原农户的生活能源消费量显著下降,但它引起的生活能源消费量下降速度低于其自身的变化速度;此外,家庭规模扩大、收入提高将使农户的生活能源消费量增加;(2)生计方式对农户能源消费模式选择的影响远高于其他因素,随着非农化水平的提高,农户选择以商品性能源为主消费模式的概率将增大,收入增加、受教育程度提高及商品性能源的可得性改善亦会如此,但生物质能源的可得性增强会降低农户选择以商品性能源为主消费模式的概率。最后,提出了优化农户生活能源消费模式的建议。     

YSK1 is activated by the Golgi matrix protein GM130 and plays a role in cell migration through its substrate 14-3-3zeta

The Golgi apparatus has long been suggested to be important for directing secretion to specific sites on the plasma membrane in response to extracellular signaling events. However, the mechanisms by which signaling events are coordinated with Golgi apparatus function remain poorly understood. Here, we identify a scaffolding function for the Golgi matrix protein GM130 that sheds light on how such signaling events may be regulated. We show that the mammalian Ste20 kinases YSK1 and MST4 target to the Golgi apparatus via the Golgi matrix protein GM130. In addition, GM130 binding activates these kinases by promoting autophosphorylation of a conserved threonine within the T-loop. Interference with YSK1 function perturbs perinuclear Golgi organization, cell migration, and invasion into type I collagen. A biochemical screen identifies 14-3-3zeta as a specific substrate for YSK1 that localizes to the Golgi apparatus, and potentially links YSK1 signaling at the Golgi apparatus with protein transport events, cell adhesion, and polarity complexes important for cell migration.