Effects of 3‐D microwell culture on initial fate specification in human embryonic stem cells

Several studies have demonstrated that three‐dimensional (3‐D) culture systems influence human embryonic stem cell (hESC) phenotypes and fate choices. However, the effect that these microenvironmental changes have on signaling pathways governing hESC behaviors is not well understood. Here, a 3‐D microwell array was used to investigate differences in activation of developmental pathways between 2‐D and 3‐D cultures of both undifferentiated hESCs and hESCs undergoing initial differentiation in embryoid bodies (EBs). An increased induction into mesoderm and endoderm and differences in expression of genes from multiple signaling pathways that regulate development, including Wnt/β‐catenin, TGF‐β superfamily, Notch, and FGF during EB‐mediated differentiation were observed in 3‐D microwells as compared with the 2‐D substrates. In undifferentiated hESCs, differences in epithelial‐mesenchymal transition phenotypes and the TGFβ/BMP pathway between cultures in 3‐D and 2‐D were also observed. These results illustrate that 3‐D culture influences multiple pathways that may regulate the differentiation trajectories of hESCs. © 2014 American Institute of Chemical Engineers AIChE J, 60: 1225–1235, 2014     

Enhancement of fibroblastic proliferation from photoreactive starch with immobilized epidermal growth factor

Carboxymethyl starch was modified by the incorporation of an azidophenyl group to prepare photoreactive starch, and characterized by Fourier transform infrared reflectance (FT‐IR), proton nuclear magnetic resonance (¹H‐NMR), and ultraviolet (UV) spectroscopy. Photo‐irradiation immobilized the Az‐starch on a polystyrene plate and it was stably retained on the surface. The protein containing immobilized Az‐starch was also immobilized on a stripe micropatterned plate. UV irradiation time and Az‐starch concentration were used to alter the physical properties of Az‐starch and consequently control the rate of epidermal growth factor (EGF) release. The Az‐starch that released growth factor was not cytotoxic to 3T3‐L1 fibroblast cells, and the immobilized EGF maintained its activity and induced cellular proliferation in vitro. These results suggest that Az‐starch could be useful as a clinical synthetic material for medical applications. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Synthesis and properties of a novel superabsorbent polymer composite from microwave irradiated waste material cultured Auricularia auricula and poly (acrylic acid‐co‐acrylamide)

A novel superabsorbent polymer composite was successfully synthesized from waste material cultured Auricularia auricula (WMCAA) and poly (acrylic acid‐co‐acrylamide) (P(AA‐co‐AM)) using microwave irradiation. Optimal synthesis conditions were determined by investigating the water absorbency of the superabsorbent composite. The effects associated with weight ratios of WMCAA, acrylamide (AM) monomers, initiators, and acrylic acid (AA) crosslinkers, as well as the degree of neutralization of AA were examined. The maximum water absorbencies were found to be 1548 g/g (distilled water) and 72 g/g (0.9% NaCl solution). Fourier transform infrared spectroscopy (FTIR) was applied to determine the molecular structure of the superabsorbent composite, and scanning electron microscopy (SEM) was used to demonstrate the characteristic compact and porous structure of the material. Further studies conducted via transmission electron microscopy (TEM) revealed the formation of a novel interpenetrating polymer network structure. Thermogravimetry/differential thermal (TG/DTG) analysis demonstrated improved thermal stability in the composite material compared with WMCAA. Additionally, high water absorption rates observed in the polymer during the swelling process indicated first‐order kinetics. The water absorption and adsorption of the superabsorbent composite were studied in a variety of fertilizer solutions, revealing an indirect relationship between water absorbing ability and fertilizer concentration. Conversely, a direct relationship was observed between absorbed fertilizer and fertilizer concentration. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 3674–3681, 2013     

Zwitterionic poly(carboxybetaine) microgels for enzyme (chymotrypsin) covalent immobilization with extended stability and activity

There is emerging evidence that biocompatible zwitterionic materials can prevent nonspecific interactions within protein systems and increase protein stability. Here, a zwitterionic microgel was synthesized from poly (carboxybetaine methyl methacrylate) (pCB) using an inverse emulsion, free radical polymerization reaction technique. The microgel was loaded with a model enzyme, α-chymotrypsin (ChT), using a post-fabrication loading technique. A reaction scheme was developed and studied for covalent immobilization of ChT within the microgel. Confocal laser microscopy studies showed that immobilized ChT (i-ChT) was distributed within the hydrogel. The enzyme-immobilized microgels showed excellent reusability (72% of its initial activity after 10 uses) and could undergo several freezing/drying/rehydration cycles while retaining enzymatic activity. The i-ChT activity, half-life, and conformational stability were studied at varying pH and temperatures with results compared to free ChT in buffer. ChT immobilized within pCB hydrogel showed increased enzymatic stability as observed by a 13°C increase in the temperature at which i-ChT loses activity compared to free ChT. Furthermore, enzyme half-life increased up to seven-fold for the pCB immobilized ChT, and the increased stability resulted in higher activity at elevated pH. The i-ChT was most active at pH of 8.5 and was partially active up to the pH of 10.2.     

Reversible control of biomaterial properties for dynamically tuning cell behavior

In the past decade, significant advances in chemistry and manufacturing have enabled the development of increasingly complex and controllable biomaterials. A key innovation is the design of dynamic biomaterials that allow for user-specified, reversible, temporal control over material properties. In this review, we provide an overview of recent advancements in reversible biomaterials, including control of stiffness, chemistry, ligand presentation, and topography. These systems have wide-ranging applications within biomedical engineering, including in vitro disease models and tissue-engineered scaffolds to guide multistep biological processes.     

In vitro and in vivo evaluation of degradability and biocompatibility of poly(p-dioxanone) hemostatic clips for laparoscopic surgery

For the clinical application of biodegradable hemostatic surgical clips in laparoscopic surgery, it is necessary to determine their degradability and biocompatibility. Herein, in vitro and in vivo studies were undertaken to evaluate the degradability and biocompatibility of bioabsorbable clips made of poly(p-dioxanone). Changes in weight loss, pull-off force, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM) of the poly(p-dioxanone) clips were determined after they were degraded in deionized water and phosphate buffer saline for the in vitro experiment and in laparoscopic models of bile duct ligation(BDL) and right gastroepiploic artery ligation(GEAL) using New Zealand white rabbits for the in vivo experiment. Changes in weight loss and pull-off force were greater in the in vivo experiment than the in vitro experiment. DSC showed the greatest variation in the degree of crystallinity of the clips degraded in deionized water. Stark differences in SEM were observed after 4 weeks of degradation both in vitro and in vivo. Furthermore, the cytocompatibility of the clips was considered satisfactory because the L929 cells could adhere to the clips and proliferate adequately in the presence of the clip extract. Biocompatibility was inferred based on the histological analysis of BDL and GEAL, no significant inflammatory responses were observed after 4 weeks of ligation.     

Measurement of the nonlinear mechanical properties of a poly(vinyl alcohol) sponge under longitudinal and circumferential loading

Poly(vinyl alcohol) sponges (P‐sponges) have been used as a potential implant material for the replacement and repair of soft tissues, including cartilage, liver, and kidney. However, the application of P‐sponges as tissue replacement materials is almost entirely bounded because of a lack of sufficient mechanical properties. In this study, we characterized the mechanical properties of a fabricated poly(vinyl alcohol) sponge (P‐sponge) under a series of longitudinal and circumferential uniaxial loadings. The nonlinear mechanical behavior of the P‐sponge was also computationally investigated with hyperelastic strain energy density functions, that is, the Ogden, Yeoh, Mooney–Rivlin, and Neo‐Hookean models. A hyperelastic constitutive model was selected to best fit the axial behavior of the sponge. The results reveal that the Young's modulus and maximum stress of the P‐sponge in the longitudinal direction were 16 and 17% greater than that in the circumferential direction, respectively. The Yeoh model, in addition, was selected to represent the nonlinear behavior of the poly(vinyl alcohol) material and could be used in future biomechanical simulations of the soft tissues. These results can be used to understand the mechanical properties of spongy materials in different loading directions. In addition, they have implications for ophthalmic and plastic surgeries and wound healing and tissue engineering purposes. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40257.     

A multi-faceted approach to analyzing glucose heat-degradants and evaluating impact to a CHO cell culture process

Glucose solutions are commonly heated before use in medical and biotech applications to maintain cleanliness, however the resulting degradants can be toxic. This study examines two approaches to evaluating the heat degradants resulting from holding a 40% glucose solution at 55°C for 5 weeks: first, chemical changes to the solution were identified and quantified via analytical testing, and second the toxicity of the heat-degraded 40% glucose solution was evaluated empirically by using it as a feed stock for a fed-batch CHO-cell-based protein therapeutic manufacturing process. Colorimetric analysis quantified a color change during heating, and liquid chromatography assays measured an increase in the concentrations of two unknown degradants along with the commonly identified glucose impurity 5-hydroxymethylfurfural (5-HMF). Solution pH decreased over time, corresponding with an increase in formic acid concentration as measured via GC–MS. Despite this, cell culture toxicity was not observed, and protein productivity and product quality were maintained.     

Influence of elastin on the properties of hybrid PCL/elastin scaffolds for tissue engineering

Developing scaffolds by combining different polymers in order to improve the properties of the bare polymers has become an extensively applied practice. Polycaprolactone (PCL) is a synthetic polyester with outstanding properties for tissue engineering (TE), although it does have certain drawbacks that can be counteracted by combining it with other biopolymers. The biopolymer elastin is an essential functional component of the dermal extracellular matrix. Therefore, the aim of this work was to produce binary systems comprising a combination of PCL (16 wt/vol%) and different concentrations of elastin (2 and 4 wt/vol%) to evaluate how the protein affects the matrix. To this end, the morphological, physicochemical, mechanical and biological properties of the aforementioned scaffolds were further characterized, observing that PCL/elastin is a suitable mixture as it improves the wettability of PCL when combined with elastin (the contact angles were reduced from 102 to ca. 70°). In addition, mixing PCL with a small quantity of elastin (2%) improved the mechanical properties of PCL-based scaffolds (Young's modulus increased from 36 to 69 MPa and the maximum stress increased from 11 to 34 MPa).     

A bio-inspired gelatin-based pH- and thermal-sensitive magnetic hydrogel for in vitro chemo/hyperthermia treatment of breast cancer cells

Gelatin (Gel)-based pH- and thermal-responsive magnetic hydrogels (MH-1 and MH-2) were designed and developed as novel drug delivery systems (DDSs) for cancer chemo/hyperthermia therapy. For this goal, Gel was functionalized with methacrylic anhydride (GelMA), and then copolymerized with (2-dimethylaminoethyl) methacrylate (DMAEMA) monomer in the presence of methacrylate-end capped magnetic nanoparticles (MNPs) as well as triethylene glycol dimethacrylate (TEGDMA; as crosslinker). Afterward, a thiol-end capped poly(N-isopropylacrylamide) (PNIPAAm-SH) was synthesized through an atom transfer radical polymerization technique, and then attached onto the hydrogel through “thiol-ene” click grafting. The preliminary performances of developed MHs for chemo/hyperthermia therapy of human breast cancer was investigated through the loading of doxorubicin hydrochloride (Dox) as an anticancer agent followed by cytotoxicity measurement of drug-loaded DDSs using MTT assay by both chemo- and chemo/hyperthermia-therapies. Owing to porous morphologies of the fabricated magnetic hydrogels according to scanning electron microscopy images and strong physicochemical interactions (e.g., hydrogen bonding) the drug loading capacities of the MH-1 and MH-2 were obtained as 72 ± 1.4 and 77 ± 1.8, respectively. The DDSs exhibited acceptable pH- and thermal-triggered drug release behaviors. The MTT assay results revealed that the combination of hyperthermia therapy and chemotherapy has synergic effect on the anticancer activities of the developed DDSs.     

Antimicrobial action and clotting time of thin,hydrated poly(vinyl alcohol)/cellulose acetate films functionalized with LL37 for prospective wound-healing applications

Poly(vinyl alcohol)/cellulose acetate (PVA/CA) films were prepared via a new method that combines principles from solvent casting and phase inversion. To guarantee some degree of flexibility, films were produced with a higher percentage of PVA compared to CA, from 90/10 to 50/50. The antimicrobial peptide (AMP) LL37 was then anchored using dopamine as a binding agent. Films were characterized in terms of functional groups, thermal stability, tensile strength, porosity, swelling, and degradation (stability in physiological media at different pHs). The antimicrobial performance of LL37 surface-modified films was tested against Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli in dynamic environment and in the presence and absence of an albumin interface. LL37 treated films demonstrated great antibacterial efficacy against the three kinds of bacteria, ≈75% inhibition for S. aureus, ≈85% for S. epidermidis, and ≈60% for E. coli, regardless of PVA/CA ratio. Presence of albumin reduced bacteria inhibition in all tested groups, most likely due to the binding of the protein molecules to the antimicrobial agents, reducing the free fraction available for bacterial killing. Films treated with LL37 accelerated clotting time (≈10 min) above vancomycin and bare surfaces, demonstrating great capacity to activate the intrinsic coagulation cascade. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48626.     

Zein nanoparticle‐embedded electrospun PVA nanofibers as wound dressing for topical delivery of anti‐inflammatory diclofenac

Bioactive wound dressings from poly(vinyl alcohol) (PVA) and zein nanoparticles (NPs) loaded with diclofenac (DLF) were prepared successfully by the single jet electrospinning method. DLF‐loaded zein NPs with an average diameter of ～228 nm were prepared using anti‐solvent precipitation method. The formulation of zein:DLF 1:1 exhibited optimum encapsulation efficiency of 47.80%. The NPs were characterized by dynamic light scattering, zeta‐potential measurement, and differential scanning calorimetry. In vitro, drug release profiles of the DLF‐loaded zein NPs, and PVA–zein NPs were also studied within 120 h and showed the release efficiency of nearly 80% from zein NPs. A more controlled release of DLF was achieved by embedding the zein NPs in the PVA nanofibers. Fourier transform infrared spectroscopy was used to analyze possible interactions between different components of the fabricated dressings. The mechanical properties of the developed dressings were also evaluated using uniaxial tensile testing. Young's modulus (E) of the dressings decreased after inclusion of zein NPs within the PVA nanofibers. Moreover, fibroblast culturing experiments proved that the composite dressings supported better cell attachment and proliferation compared to PVA nanofibers, by exhibiting moderate hydrophilicity. The results suggested that the electrospun composite dressing of PVA nanofibers and zein NPs is a promising topical drug‐delivery system and have a great potential for wound healing application. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46643.     

Preparation and characterization of poly(ethylene glycol) stabilized nano silver particles by a mechanochemical assisted ball mill process

Nano silver particles with an average mean crystallite size of between 10 and 12 nm were synthesized from different molecular weights of poly(ethylene glycol) as a stabilizing agents, through solid state oxidation of silver nitrate using a higher energy planetary ball mill. Ultraviolet‐Visible spectra were used to confirm the synthesis of nano silver particles. The surface plasmon resonance bands were observed around 410 nm. Fourier transformed infrared spectrum, X‐ray diffraction, and transmission electronic microscopy techniques were used to characterize the nano silver particles synthesized. Thermal stability was determined using thermogravimetic analysis and the elemental composition of the sample was determined by energy dispersive X‐ray analysis. The nano silver particles synthesized, exhibited very good antibacterial activity against Gram‐positive bacteria (Bacillus) and Gram‐negative bacteria (Pseudomonas aeruginosa). Based on the obtained results, it was additionally explored that the size and the stabilization of the nano silver particles synthesized, strongly depend on the molecular weight of poly(ethylene glycol). © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43027.     

生物工程专业化工原理课程教学改革探索与思考

化工原理是生物工程专业的基础技术课,它在基础课与专业课之间,起着承前启后,由＂理＂及＂工＂的桥梁作用。主要任务是研究生物过程中常见的单元操作的基本原理,典型设备的构造及工艺尺寸的计算或选型,指出强化生产过程的途径和提高设备能力,效率以及降低成本的措施。本文结合生物工程专业的特点,对化工原理课程的教学进行了探索,收到了较好的效果。     

Immobilization of multicopper oxidase from Pyrobaculum aerophilum onto an electrospun-aligned single-walled carbon nanotube surface via a carbon-nanotube-binding peptide for biocathode

Biofuel cells (BFCs) that produce electrical energy from organic resources through enzymatic reactions have been attracting significant attention. Owing to the high electrical conductivity of carbon nanotubes (CNTs), their modification on the electrode surface of a BFC is expected to increase the current, and their high specific surface area may be useful in increasing the power output. Previously, we constructed a biocathode by immobilizing multicopper oxidase from Pyrobaculum aerophilum (McoP) with a carbon nanotube binding peptide (CBP) sequence on the CNTs. This resulted in higher current densities than when using enzymes without CBP sequences. However, owing to the randomly stacked CNTs on the surface of the electrodes, their conductive properties were impaired and performance as biocathodes was poor. Herein, we constructed a biocathode in which single-walled CNTs (SWCNTs) were oriented one-dimensionally and McoP is immobilized on the surface of an SWNCT via CBP. The current density was successfully increased by two-fold by orienting the CNTs and orienting and immobilizing McoP on their surfaces. This technology provides insights into the development of biodevices with controlled orientation of both the SWCNTs and enzymes immobilized on their surfaces.     

Chitosan-based fiber-sponge materials as a promising tool for microalgae harvesting from Lake Baikal

Mass eutrophication of microalgae and cyanobacteria is observed in Lake Baikal in the past decade. In this paper, the concept of replaceable adsorption filter material based on chitosan flocculant filler and chlorinated polyvinyl chloride polymer nonwoven material are proposed. Functional and mechanical properties and morphology of the material are investigated depending on a packing density and a degree of chitosan filling. The introduction of 45% chitosan increases the Young's modulus up to 10 times, and it makes the material more rigid in 2.8 times. The high efficiency of sorption and growth inhibition of cumulative biomass culture was shown. The biomass source is taken from the coast of Barguzinsky Bay of Lake Baikal. Dominant species is microalgae of Scenedesmus genus.     

Confirmation of the predicted source of a slow folding reaction: proline 8 of bovine pancreatic trypsin inhibitor

A major question in protein structural analysis concerns theapplicability of results from model systems to other proteins.Theoretical approaches seem the best manner of transferringinformation from one system to another, but their accuracy inthe model systems must first be tested with results from experiment.Since bovine pancreatic trypsin inhibitor (BPTI) is a modelsystem for the evaluation of energy minimization and moleculardynamics routines, we can use folding and stability measurementsto examine the reliability of these methods. All two-disulfidemutants of BPTI investigated thus far have two very slow foldingreactions which have characteristics of proline isomerization.These reactions may occur because the non-native cis form oftwo of the four prolines in BPTI significantly destabilizesthe folded state of the protein. Previous energy minimizationstudies of wild-type BPTI suggested that the cis form of Pro8was the most destabilizing of the four prolines [Levitt,M. (1981)J. Mol. Biol., 145, 251–263]. In this paper, we show thatmutation of Pro8 - Gln in the two-disulfide bond mutant Val30–Ala51results in a loss of the slowest folding reaction, consistentwith Levitt's prediction.     

重组人骨形态发生蛋白-2的制备及成骨活性表征

骨形态发生蛋白-2(BMP-2)是重要的骨诱导生长因子,是提高骨修复材料活性和临床骨修复效果的有效手段和关键物质。由于BMP-2在体内含量低,依靠从动物体内提取难以满足临床需求。本文研究满足临床需求的BMP-2的制备方法,并评价其生物活性。采用密码子优化方法,并通过进一步更换其中部分核苷酸编码,得到优化的hBMP-2基因的DNA序列,制备大肠杆菌rhBMP-2菌株,通过发酵及工艺优化,获得BMP包涵体,经分离纯化与复性,制备出高纯度的rhBMP-2。测定C2C12细胞的碱性磷酸酶(ALP)活性来表征单倍体和二倍体BMP-2的成骨活性,发现二倍体rhBMP-2的成骨活性明显高于单倍体rhBMP-2,并且随着BMP-2浓度增加,碱性磷酸酶活性上升。体内动物异位成骨实验发现rhBMP-2肌带植入小鼠体内3周后,取出的异位骨颗粒鲜艳饱满,骨结构完整;HE切片和Masson三色切片都显示出良好的异位成骨效果。此方法制备的rhBMP-2具有良好的诱导成骨分化能力,可用于骨组织修复,满足临床需要。