Hydrogels with Dynamically Controllable Mechanics and Biochemistry for 3D Cell Culture Platforms

Many cell-matrix interaction studies have proved that dynamic changes in the extracellular matrix(ECM)are crucial to maintain cellular properties and behaviors.Thus,developing materials that can recapitulate the dynamic attributes of the ECM is highly desired for threedimensional(3 D)cell culture platforms.To this end,we sought to develop a hydrogel system that would enable dynamic and reversible turning of its mechanical and biochemical properties,thus facilitating the control of cell culture to imitate the natural ECM.Herein,a hydrogel with dynamic mechanics and a biochemistry based on an addition-fragmentation chain transfer(AFCT)reaction was constructed.Thiol-modified hyaluronic acid(HA)and allyl sulfide-modifiedε-poly-L-lysine(EPL)were synthesized to form hydrogels,which were non-swellable and biocompatible.The reversible modulus of the hydrogel was first achieved through the AFCT reaction;the modulus can also be regulated stepwise by changing the dose of UVA irradiation.Dynamic patterning of fluorescent markers in the hydrogel was also realized.Therefore,this dynamically controllable hydrogel has great potential as a 3 D cell culture platform for tissue engineering applications.     

Size‐Controlled Fabrication of Polyaniline Microfibers Based on 3D Hydrodynamic Focusing Approach

Owing to the relatively high conductivity and unique redox behavior, polyaniline (PANI) has been one of the most technologically promising conducting polymers. Although various methodologies have been developed, fabrication of PANI microfibers has been a challenging task owing to the poor solubility in most organic solvents. By taking advantage of a microfluidic technology and organic soluble acid labile t‐Boc‐protected PANI (t‐Boc‐PANI) as the conducting polymer precursor, fabrication of PANI microfibers in a size‐controlled manner is possible. Introduction of a THF solution containing t‐Boc‐PANI, and dodecylbenzenesulfonic acid (DBSA) as a core flow, and water as a sheath flow into a microfluidic channel with a 3D hydrodynamic focusing effect results in crystallization of the polymer fiber. By changing the flow rate, linear PANI microfibers that range from 16.2 to 39.4 μm in diameter are readily obtained.

     

Acrylic‐Acid‐Functionalized PolyHIPE Scaffolds for Use in 3D Cell Culture

This study describes the development of a functional porous polymer for use as a scaffold to support 3D hepatocyte culture. A high internal phase emulsion (HIPE) is prepared containing the monomers styrene (STY), divinylbenzene (DVB), and 2‐ethylhexyl acrylate (EHA) in the external oil phase and the monomer acrylic acid (Aa) in the internal aqueous phase. Upon thermal polymerization with azobisisobutyronitrile (AIBN), the resulting porous polymer (polyHIPE) is found to have an open‐cell morphology and a porosity of 89%, both suitable characteristics for 3D cell scaffold applications. X‐ray photo­electron spectroscopy reveals that the polyHIPE surface contained 7.5% carboxylic acid functionality, providing a useful substrate for subsequent surface modifications and bio‐conjugations. Initial bio‐compatibility assessments with human hepatocytes show that the acid functionality does not have any detrimental effect on cell adhesion. It is therefore believed that this material can be a useful precursor scaffold towards 3D substrates that offer tailored surface functionality for enhanced cell adhesion.

     

Size‐Controlled Fabrication of Polydiacetylene‐Embedded Microfibers on a Microfluidic Chip

A microfluidic technique was employed to fabricate polydiacetylene (PDA)‐embedded hydrogel microfibers. By taking advantage of calcium ion‐induced insoluble hydrogel formation, supramolecularly assembled diacetylene (DA)‐surfactant complexes were successfully immobilized in the calcium alginate fibers. Thus, instantaneous microfiber formation was observed when the core flow of DA supramolecules‐containing alginate solution met the sheath flow of calcium ions. UV irradiation of the resulting fibers afforded blue colored PDAs, and the formation of a conjugated polymer was confirmed by heat‐induced phase transition and by Raman spectroscopy. By adjusting the core and sheath flow rates, PDA‐embedded hydrogel fibers of various sizes were obtained.     

Comprehensive Examination of Mechanical and Diffusional Effects on Cell Behavior Using a Decoupled 3D Hydrogel System

Hydrogels possess several physical and chemical properties suitable for engineering cellular environments for biomedical applications. Despite recent advances in hydrogel systems for cell culture, it is still a significant challenge to independently control the mechanical and diffusional properties of hydrogels, both of which are well known to influence various cell behaviors when using hydrogels as 3D cell culture systems. Controlling the crosslinking density of a hydrogel system to tune the mechanical properties inevitably affects their diffusional properties, as the crosslinking density and diffusion are often inversely correlated. In this study, a polymeric crosslinker is demonstrated that allows for the adjustment of the degree of substitution of reactive functional groups. By using this polymeric crosslinker, the rigidity of the resulting hydrogel is controlled in a wide range without changing the polymer concentration. Furthermore, their diffusional properties, as characterized by their swelling ratios, pore diameters, and drug release rates, are not significantly affected by the changes in the degree of substitution. 3D cell studies using this hydrogel system successfully demonstrate the varying effects of mechanical properties on different cell types, whereas those in a conventional hydrogel system are more significantly influenced by changes in diffusional properties.     

可用于3D肝细胞培养的甘草次酸修饰海藻酸钠凝胶球的制备

制备了叔胺改性甘草次酸[GA-N(CH₃)₂]修饰的海藻酸钠[ALG-GA-N(CH₃)₂], 并在温敏性琼脂糖的辅助作用下, 利用微流体技术获得了高通量、 单分散且粒径可控的ALG-GA-N(CH₃)₂微凝胶. 考察了Span 80含量、 疏水配体取代度、 样品浓度和水/油相流速对微液滴制备的影响. 研究结果表明, 叔胺基改性可显著改善甘草次酸的亲水性; 在Span 80质量分数为2.0%, 疏水配体取代度小于12%, 样品浓度小于15 mg/mL, 水相流速为1.5 mL/h, 油相流速为6 mL/h条件下, 可获得高通量、 单分散及粒径为200 μm的适用于细胞包封培养的微凝胶球. 同时提供了一种三维培养肝细胞的新方法, 为其在组织工程中的应用奠定了基础.     

纤维状TiO2／Bi2O3光催化剂的制备及其光催化性能

研究了SrCo0．7Fe0．2Mo0．103-δ（SCFM）材料的相组成、微观结构、热膨胀系数、氧渗透性能和化学稳定性,其结果和文献中的SrCo0．8Fe0.2O3-δ（SCF）做了对比．通过EDTA-citric混合方法成功获得了纯相SCFM材料．SCFM材料在500-1050℃显示出比SCF材料更低的热膨胀系数（24×10^-6-29×10^-6／K）,表明其具有一种更稳定的结构,尽管由于Mo掺杂造成其透氧率比SCF材料低,但是SCFM的透氧率仍然维持在一个较高水平．证实SCF中的Mo掺杂能够阻止晶格中的有序-无序转变,提高了其在CO2下的化学稳定性．     

Physical Models from Physical Templates Using Biocompatible Liquid Crystal Elastomers as Morphologically Programmable Inks For 3D Printing

Advanced manufacturing has received considerable attention as a tool for the fabrication of cell scaffolds however, finding ideal biocompatible and biodegradable materials that fit the correct parameters for 3D printing and guide cells to align remain a challenge. Herein, a photocrosslinkable smectic-A (Sm-A) liquid crystal elastomer (LCE) designed for 3D printing is presented, that promotes cell proliferation but most importantly induces cell anisotropy. The LCE-based bio-ink allows the 3D duplication of a highly complex brain structure generated from an animal model. Vascular tissue models are generated from fluorescently stained mouse tissue spatially imaged using confocal microscopy and subsequently processed to create a digital 3D model suitable for printing. The 3D structure is reproduced using a Digital Light Processing (DLP) stereolithography (SLA) desktop 3D printer. Synchrotron Small-Angle X-ray Diffraction (SAXD) data reveal a strong alignment of the LCE layering within the struts of the printed 3D scaffold. The resultant anisotropy of the LCE struts is then shown to direct cell growth. This study offers a simple approach to produce model tissues built within hours that promote cellular alignment.     

一种用于FDM型3D打印的改性PBT

以对苯二甲酸(PTA)、间苯二甲酸(PIA)、癸二酸(SA)和1,4-丁二醇(BDO)为原料,通过直接酯化和减压缩聚的方法制备了一种适合熔融沉积打印(FDM)型3D打印的改性聚对苯二甲酸丁二醇酯(PBT)——聚对苯二甲酸间苯二甲酸癸二酸丁二醇酯(PBTIS)。采用核磁共振氢谱(1 H-NMR)、凝胶渗透色谱法(GPC)、差示扫描量热法(DSC)、黏度计和熔体仪、万能电子拉力机和冲击试验机分别研究了其热性能、流变性能和力学性能。研究表明:当n(PTA)∶n(PIA)=7∶3,SA的物质的量分数为3%~5%时,PBTIS具有合适的熔点、良好的拉伸强度和弯曲强度及悬梁缺口冲击强度等力学性能。用桌面拉丝挤出机将PBTIS样品制成卷材并用3D打印机打印,结果表明其可以流畅地打印出所设计的三维物件。     

Enhanced Power Density of Alcohol Biofuel Cell by Polymer-assisted Crosslinks of 3D Graphene on Carbon Paper as the Bioanode

Herein, we successfully construct the 3D biocompatible graphene through crosslinking 2D graphene nanosheet onto carbon fiber paper with poly(diallyldimethylammonium chloride) (PDDA) as anode of the alcohol biofuel cell. Compared with the bioanode without 3D graphene, the current density and output power of PDDA-graphene-ADH bioanode is increased by 23 % and 41 % at a high concentration of ethanol at pH 8.9, suggesting the stabilization role of graphene in enzyme loading. The study provides us a deep analysis on structures and performances of the bioanode incl. electrochemistry, X-ray photoelectron spectra, and atomic force microscopy images, which is significant to develop the new methods to construct 3D porous electrodes in energy conversion device.     

Development and Characterization of an Orodispersible Film for Vitamin D3 Supplementation

Vitamin D plays a crucial and very well-known role in regulation of calcium homeostasis and bone metabolism and mineralization. However, a huge and more recent body of evidence supports the positive influence of vitamin D on the regulation of immune response, ranging from protection against respiratory tract infections to prevention and management of asthma. Nevertheless, vitamin D deficiency is a very common condition and there is an increasing need for suitable products for proper supplementation, allowing good compliance also in specific populations. Orally disintegrating tablets (ODT) were first developed to overcome the difficulty experienced by pediatric and geriatric patients of swallowing traditional oral dosage forms and, recently, orodispersible films (ODF) are gaining popularity as novel dosage form for assuming active pharmaceutical ingredients, vitamins, and ingredients for food supplements. This study describes a 2000 IU Vitamin D3 ODF for daily intake, consisting of hydrophilic polymers and suitable excipients, manufactured by film-casting process. Elongation-at-break (E%), Young’s modulus (Y), and tensile strength (TS) were investigated using a dynamometer. Chemical stability was evaluated assaying the vitamin D3 in the films stored at different environmental conditions. In addition, in vitro disintegration and dissolution studies were performed. Correlation existed between the mechanical properties of the film and the residual water, acting as plasticizer. The stability study showed that vitamin D3 assay was ≥90% also after 3 months at 40 °C. The film disintegrated in less than 1 min and the vitamin D3 released was ≥75% after 15 min. An ODF with suitable properties can be manufactured and used as innovative dosage form for vitamin D3 food supplements.     

自组装短肽GFS-2构建新型三维细胞培养支架材料

典型的自组装短肽在水中可形成稳定的β-sheet二级结构,在生理性环境下则能够形成稳定的纳米纤维,可进一步形成含水量达99%的水凝胶,具有高纯度、可降解及无免疫反应等突出优点,能模拟生物体内的三维基质环境而作为细胞三维培养的新型生物材料。本文主要采用了圆二色谱仪、原子力显微镜、倒置显微镜等探究新型短肽GFS-2的自组装性能及其在细胞三维培养中的应用。此研究可能会启发设计更多的新型自组装短肽服务于化学、生物材料、医学工程等领域。     

Enhanced Cell Penetration and Pluripotency Maintenance of hiPSCs in 3D Natural Chitosan Scaffolds

Human-induced pluripotent stem cells (hiPSCs) cultured in 3D matrices hold great promise in disease modeling, drug discovery, and tissue regeneration. Uniform cell distribution in a 3D structure is critical to the growth and function of hiPSCs, yet cell seeding in 3D matrices often remains superficial, leading to limited cell proliferation and compromised pluripotency. Here, an approach to improve cell penetration depth of hiPSCs in 3D scaffolds modified with hiPSCs conditioned medium (CM) is reported. It is shown that extracellular matrix components are successfully deposited onto the scaffold wall surface after CM treatment and promoted homogeneous cell adhesion during initial seeding. Compared to plain, unmodified scaffolds, the CM treated scaffold improves spatial cell distribution uniformity and upregulates pluripotency markers. Notably, the expression of 29 genes associated with 11 signaling pathways participated in the pluripotency maintenance of hiPSCs exhibits >2-fold change in hiPSCs grown in the CM treated scaffolds than 2D counterparts, demonstrating that CM treated scaffolds can support a more primitive and undifferentiated phenotype of hiPSCs. This study introduces a simple and effective method to enhance cell penetration and maintain cell pluripotency in 3D matrices.     

The influence of grafted cellulose nanofibers and postextrusion annealing treatment on selected properties of poly(lactic acid) filaments for 3D printing

l ‐lactide monomers were grafted onto cellulose nanofibers (CNFs) via ring‐opening polymerization, forming poly(lactic acid) grafted cellulose nanofibers (PLA‐g‐CNFs). PLA‐g‐CNFs and pristine PLA were then blended in chloroform and dried to prepare a master batch. PLA‐g‐CNFs/PLA composite filaments targeted for 3D printing were produced by compounding the master batch in PLA matrix and melt extrusion. The as‐extruded composite filaments were subsequently thermal annealed in a conventional oven, and their morphological, thermal, and mechanical properties were evaluated. PLA was successfully grafted on the surface of CNFs as demonstrated by elemental analysis, and the concentration of grafted PLA was estimated to be 33 wt %. The grafted PLA were highly crystallized, contributing to the growth of crystalline regions of PLA matrix. The incorporation of PLA‐g‐CNFs improved storage modulus of the composite filaments in both low temperature glassy state and high temperature rubbery state. Postextrusion annealing treatment led to 28 and 63% increases for tensile modulus and strength of the filaments, respectively. Simulated Young's moduli from the Halpin‐Tsai and Krenchel models were found comparable with the experimental values. The formed composite filaments are suitable for use in 3D printing. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2017 , 55, 847–855     

3D printing to innovate biopolymer materials for demanding applications: A review

Biopolymers are widely available, low-/nontoxic, biodegradable, biocompatible, chemically versatile, and inherently functional, making them highly potential for a broad range of applications such as biomedicine, food, textile, and cosmetics. 3D printing (3DP) is capable of fabricating some customized, complex material structures composed of single or multiple material constituents that cannot be achieved by conventional methodologies (e.g. internal structures design); thus, 3DP can greatly expand the application of biopolymer materials. This review presents a comprehensive survey of the latest literature in 3DP technology for materials from biopolymers such as polysaccharides and proteins. The most commonly used 3DP techniques (i.e. inkjet printing, extrusion-based printing, stereolithography, selective laser sintering, and binder jetting) in biomedical and food fields are discussed. Critical factors affecting the quality and accuracy of 3D-printed constructs, including rheological characteristics, printing parameters (e.g. printing rate, and nozzle diameter, movement rate, and height), and post-printing processes (e.g. baking, drying, and crosslinking) are analyzed. The properties and the emerging applications of 3D-printed biopolymer materials in biomedical, food, and even wider applications (e.g. wastewater treatment and sensing) are summarized and evaluated. Finally, challenges and future perspectives are discussed. This review can provide insights into the development of new biopolymer-based inks and new biopolymer-based 3D-printed materials with enhanced properties and functionality.     

Biofunctional rapid prototyping for tissue-engineering applications: 3D bioplotting versus 3D printing

Two important rapid-prototyping technologies (3D Printing and 3D Bioplotting) were compared with respect to the computer-aided design and free-form fabrication of biodegradable polyurethane scaffolds meeting the demands of tissue-engineering applications. Aliphatic polyurethanes were based on lysine ethyl ester diisocyanate and isophorone diisocyanate. Layer-by-layer construction of the scaffolds was performed by 3D Printing, that is, bonding together starch particles followed by infiltration and partial crosslinking of starch with lysine ethyl ester diisocyanate. Alternatively, the 3D Bioplotting process permitted three-dimensional dispensing and reactive processing of oligoetherurethanes derived from isophorone diisocyanate, oligoethylene oxide, and glycerol. The scaffolds were characterized with X-ray microtomography, scanning electron microscopy, and mechanical testing. Osteoblast-like cells were seeded on such scaffolds to demonstrate their potential in tissue engineering. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 624–638, 2004     

三维多孔碳纤维/聚乳酸/壳聚糖复合支架材料的体外细胞相容性评价

利用溶液共混法以及冷冻干燥法制备了三维多孔碳纤维/聚乳酸/壳聚糖(CF/PLA/CS)复合生物支架材料,通过相差显微镜和扫描电子显微镜检测了鼠骨髓基质细胞（BMSCs）与该材料的生物相容性,得到了MTT生长曲线,评价了材料的毒性。 结果表明,以实验组材料的浸提液培养细胞,8 d后细胞开始连片生长,没有观察到细胞变形、坏死现象;在实验组材料上培养4 d后细胞的SEM图像显示,细胞形貌正常,并已开始向孔隙深部生长;MTT法绘制的增值曲线表明,培养4 d后实验组的细胞增殖速度高出空白对照组30%。 以上细胞形态学观察法和细胞增殖法评价结果表明,三维多孔 CF/PLA/CS复合材料没有细胞毒性,并对细胞有良好的粘附、增殖能力,有望成为骨修复材料。     

Micro‐Engineered 3D Scaffolds for Cell Culture Studies

Cells in physiological 3D environments differ considerably in morphology and differentiation from those in 2D tissue culture. Naturally derived polymer systems are frequently used to study cells in 3D. These 3D matrices are complex with respect to their chemical composition, mechanical properties, and geometry. Therefore, there is a demand for well‐defined 3D scaffolds to systematically investigate cell behavior in 3D. Here, fabrication techniques, materials, architectures, biochemical functionalizations, and mechanical properties of 3D scaffolds are discussed. In particular, work focusing on single cells and small cell assemblies grown in tailored synthetic 3D scaffolds fabricated by computer‐based techniques are reviewed and the influence of these environments on cell behavior is evaluated.

     

Chromatographic Processing Scheme for Continuous Harvesting of rec-Prourokinase from Serum Free Medium Cell Cultures

Continuous perfusion cell culture, using albumin containing medium, offers the potential advantages of higher recombinant Prourokinase (r-ProUK) yields, higher initial product purity and increased throughput compared to batch culture technology using medium supplemented with fetal bovine serum. We have characterized the production of r-ProUK in medium supplemented with a lipid rich bovine serum albumin (Albumax) in a perfusion system. The results of these studies showed that it was necessary to modify the r-ProUK batch recovery scheme to process r-ProUK from a perfusion system. To accommodate large volumes of perfusate harvested over a ten to fourteen day production cycle, cation exchange and hydrophobic interaction chromatography (HIC) resins were identified that had increased product binding capacity, better flow characteristics and wider pH ranges which allowed caustic cleaning. The mobile phase composition, pH and ionic strength were modified to improve r-ProUK yields from the identified resins, and procedures were developed to eliminate r-ProUK degradation products. Strategies were defined for processing continuous harvest, which contained four to seven times the amount of r-ProUK of batch harvests.