Crosslinking Conditions Research Articles

Preparation and Characterization of Biomimetic β-Lens Crystallins Using Single-Chain Polymeric Nanoparticles.

Presbyopia, the inability to focus at arm's length, and cataracts that cloud vision are associated primarily with changes in the mechanical and optical properties of the lens. The optical properties, particularly the refractive index, of the human lens originate from the cytoplasm of the lens fiber, which contains a highly concentrated solution (∼40%) of globular proteins referred to as α, β, and γ crystallins, of which β is the most abundant. In this study, we focus on the synthesis and characterization of a β-crystallin biomimetic in an effort to understand and develop treatments for presbyopia and cataract. Polyacrylamide was used as a protein analogue. The side chains were endowed with aromatic and acidic functionality. Acrylic acid was incorporated into the copolymer and cross-linked with diamines to form nanoparticles. The composition and cross-linking condition of the biomimetic copolymers were optimized to match the hydrodynamic radius (Rh), refractive index, size, density, and intrinsic and dynamic viscosities with those of βhigh lens crystallins. The refractive indices and densities of the nanoparticles' dispersion at different concentrations matched that of βhigh lens crystallins, and the viscosity of the nanoparticles approached that of βhigh lens crystallins. The biocompatibility findings for primary porcine retinal pigment epithelial (ppRPE) cells and porcine lens epithelial (pLE) cells showed both cell types tolerated up to 30 mg/mL of nanoparticles. These materials have the potential for use as replacements for the crystallins in developing an accommodating intraocular lens nanocomposite hydrogel that closely replicates the natural autofocusing ability of the original.

Molecular forms of ruminant BMP15 and GDF9 and putative interactions with receptors.

Bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-secreted factors with demonstrable effects on ovarian follicular development and ovulation rate. However, the molecular forms of BMP15 and GDF9 produced by oocytes remain unclear. The aims herein, using Western blotting (WB) procedures with specific monoclonal antibodies (mabs), were to identify the molecular forms of BMP15 and GDF9 synthesised and secreted by isolated ovine (o) and bovine (b) oocytes in vitro The mabs were known to recognise the biological forms of BMP15 or GDF9 since they had previously been shown to inhibit their bioactivities in vitro and in vivo Using recombinant variants of oBMP15 and oGDF9, including a cysteine mutant form of oBMP15 (S356C) and a human (h) BMP15:GDF9 heterodimer (cumulin), it was established that the mabs were able to identify monomeric, dimeric, promature and higher-molecular-weight forms of BMP15 and GDF9 and cumulin (GDF9 mab only). After using non-reducing, reducing and reducing + cross-linking conditions, the major oocyte-secreted forms of o and b BMP15 and GDF9 were the cleaved and uncleaved monomeric forms of the promature proteins. There was no evidence for dimeric or heterodimeric forms of either mature BMP15 or GDF9. From in silico modelling studies using transforming growth factor beta (TGFB), activin or BMP crystal templates, and both present and previously published data, a model is proposed to illustrate how the monomeric forms of BMP15 and GDF9 may interact with their type II and type I cell-surface receptors to initiate the synergistic actions of these growth factors.

Robust and fire retardant borate-crosslinked poly (vinyl alcohol)/montmorillonite aerogel via melt-crosslink

A highly robust and flame retardant borate-crosslinked poly (vinyl alcohol)/montmorillonite aerogel is fabricated through a novel melt-crosslink method. In this way, the PVA/MMT hydrogel was prepared by immersing the frozen PVA/MMT sol into a borax solution, inducing crosslinking between diols of PVA and borate exactly during the ice melt. As a result, rearrangement of the polymer chains and subsequent expansion result in finer and more compact network. The corresponding aerogel is obtained by freeze-drying. The optimal crosslinking conditions, including the borax concentration and crosslinking time, are determined by rheology of the hydrogels. This method provides a new idea for the preparation of crosslinked aerogels and improvement of mechanical and flame retardant properties simultaneously. The mechanical strength of the crosslinked aerogel is relatively high among the existing fire safety polymer/nanoclay composite aerogels with 30%–60% loading of inorganic matters at a low density level. The flame retardant properties are indicated by significant improvements of limiting oxygen index (LOI) to a value of 27.6% and achievement of UL-94 V-0 rating and remarkable decrease of the fire indexes in cone calorimetry (CC) test. The combustion behaviors are further studied by the results of SEM, energy dispersive X-ray spectrometer (EDX) and X-ray photoelectron spectroscopy (XPS).

High-performance composite hollow fiber membrane for flue gas and air separations

A defect-free membrane with a very high gas permeance is greatly attractive to both academia and industry. Thin film composite (TFC) membranes are promising candidates. However, it is always challenging to have a reproducible and up-scalable method to fulfill the needs. Herein, we report a novel and straightforward strategy to fabricate a high-performance hollow fiber composite membrane using a crosslinked polydimethylsiloxane (PDMS) with a high inherent viscosity obtained from a novel post-crosslinking method. The evolution of inherent viscosity with various cross-linking conditions and substrate morphology from different spinning conditions have been investigated. The resultant defect-free composite membrane shows excellent O2 and CO2 permeances higher than 1000 and 5000GPU, respectively; while the corresponding selectivities of O2/N2 and CO2/N2 are about 2 and 11, respectively. The newly developed methods may provide useful insights to fabricate next-generation high-performance composite membranes for gas separation.

Abstract 2114: Targeting invasive and drug-resistant PDAC with photodynamic therapy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is associated with an extensive desmoplastic reaction and a characteristic extracellular matrix (ECM)-rich stromal microenvironment which is a major determinant of disease progression and therapeutic response. Using 3D cell culture models of PDAC, we recently showed that an invasion-promoting ECM composition leads to differential response to chemotherapy agents and photodynamic therapy (PDT), in which cytotoxic response is triggered by light activation of a photosensitizing agent. In particular, ECM-infiltrating populations with characteristic mesenchymal phenotype display a marked increase in sensitivity to PDT (using the photosensitizer verteporfin), while the same cells exhibit increased chemoresistance. Here, motivated by these findings we systematically disentangle influences associated with the physical and biochemical properties of ECM as regulators of PDAC growth behavior and treatment response. In 3D tumor spheroids transplanted into invasion-promoting type 1 collagen gels, we use riboflavin-mediated photocrosslinking as a tool to regulate ECM stiffness (independent of its concentration), combined with high-content imaging to co-register invasive progression and differential response to chemotherapy and PDT. Increased extent of photocrosslinking correlates with increased collagen stiffness as confirmed by bulk oscillatory rheology, and is associated with a decrease in the invasive velocity (migration speed directed away from the primary transplanted spheroid) of ECM-infiltrating PDAC cells. Preliminary comparisons of PDT and oxaliplatin chemotherapy in low and high collagen crosslinking conditions show that PDT is significantly more effective than oxaliplatin in ECM-invading populations regardless of collagen stiffness. We also compare oxaliplatin chemotherapy and PDT before and after PDAC invasion into Matrigel ECM, and find that PDT efficacy is only enhanced after extensive invasion into the surrounding matrix. These results support the efficacy of PDT in targeting locally invasive PDAC while stressing the importance of elucidating how both biochemical and biophysical ECM interactions regulate therapeutic susceptibilities. Citation Format: Gwendolyn Cramer, Seyedehrojin Jafari, Hamid El-Hamidi, Dustin Jones, Jonathan Celli. Targeting invasive and drug-resistant PDAC with photodynamic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2114. doi:10.1158/1538-7445.AM2017-2114

Development and investigation of mixed-matrix PVA-fullerenol membranes for acetic acid dehydration by pervaporation

Abstract This study focuses on the development of a green pervaporation (PV) process for dehydrating a wide range of commercially significant mixtures such as concentrated (and corrosive) acetic acid blends using mixed-matrix membranes based on polyvinyl alcohol (PVA) cross-linked by low-hydroxylated fullerenol (C 60 (OH) 12 ). The effect of C 60 (OH) 12 and various conditions of physical cross-linking on the structure and internal morphology of composite membranes was investigated by X-ray diffraction and scanning electron microscopy, and the stability and transport properties of the developed membranes were investigated for acetic acid dehydration at a wide range of feed water contents (5–30 wt.%) and temperatures (25–60 °C). The supported mixed-matrix membranes exhibited high permeances and water selectivities, as well as good stability toward feed mixtures, with the best transport properties obtained for a C 60 (OH) 12 (5 wt.%)-PVA supported composite membrane thermally treated at 140 °C for 420 min.

To prepare the collagen‐based artificial cornea with improved mechanical and biological property by ultraviolet‐A/riboflavin crosslinking

ABSTRACTIn the present study, we optimized the ultraviolet‐A (UVA)/riboflavin crosslinking for the collagen‐based artificial cornea for the first time to resolve the lack of donor cornea for corneal disease. We found that the crosslinking conditions, including irradiation time and irradiation intensity, played important roles on the property of the sample. At the optimal condition (with the irradiation time of 30 min and the irradiation intensity of 5 mW/cm2), the sample exhibited excellent crosslink degree and mechanical property while keeping good light transmittance. Meanwhile, the sample showed improved enzyme tolerance capacity and thermal stability. This developed artificial cornea also had good biocompatibility to human corneal epithelial cells in vitro. In vivo lamellar keratoplasty results showed that the developed sample could promote complete epithelialization in about 4 weeks, and the transparency is restored quickly in the first 6 weeks. Corneal rejection reaction and keratoconus are not observed. This optimized UVA/riboflavin crosslinking method would have great potential for collagen‐based artificial cornea in clinic. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 45226.

A highly adhesive and naturally derived sealant.

Conventional surgical techniques to seal and repair defects in highly stressed elastic tissues are insufficient. Therefore, this study aimed to engineer an inexpensive, highly adhesive, biocompatible, and biodegradable sealant based on a modified and naturally derived biopolymer, gelatin methacryloyl (GelMA). We tuned the degree of gelatin modification, prepolymer concentration, photoinitiator concentration, and crosslinking conditions to optimize the physical properties and adhesion of the photocrosslinked GelMA sealants. Following ASTM standard tests that target wound closure strength, shear resistance, and burst pressure, GelMA sealant was shown to exhibit adhesive properties that were superior to clinically used fibrin- and poly(ethylene glycol)-based glues. Chronic invivo experiments in small as well as translational large animal models proved GelMA to effectively seal large lung leakages without the need for sutures or staples, presenting improved performance as compared to fibrin glue, poly(ethylene glycol) glue and sutures only. Furthermore, high biocompatibility of GelMA sealant was observed, as evidenced by a low inflammatory host response and fast invivo degradation while allowing for adequate wound healing at the same time. Combining these results with the low costs, ease of synthesis and application of the material, GelMA sealant is envisioned to be commercialized not only as a sealant to stop air leakages, but also as a biocompatible and biodegradable hydrogel to support lung tissue regeneration.

Thermo-rheological properties of coal-tar pitch modified with phenol-formaldehyde resin

Test results of changing the thermo-rheological properties of coal-tar pitch under influence of phenol-formaldehyde resin are presented in this research work. Influence of modifying agent amount and crosslinking conditions (time) of resin in bitumen was evaluated. Tests were carried out using the rheometer in oscillation mode. They enabled the evaluation of changes in bitumen viscous and elastic properties and draw the conclusions about changes in bitumen structure on that basis. Compositions of the coal-tar pitch with phenol-formaldehyde resin show increased thermal and mechanical resistance. Furthermore, phenol-formaldehyde resin has an effect on increase in the coal-tar pitch viscosity. It probably results from the increased content of components of higher average molecular mass in the pitch and lower solubility in organic solvents.

Improvement of mechanical properties and antibacterial activity of crosslinked electrospun chitosan/poly (ethylene oxide) nanofibers

Abstract In this study conditions for green crosslinking with citric acid of chitosan/PEO (polyethylene oxide) nanofibers were evaluated. The thermal in situ crosslinking enabled penetration of crosslinking agent into the matrix providing an improvement of antibacterial activity, thermal stability and mechanical properties of the prepared material. With an increase of temperature above 80 °C antibacterial activity against Staphylococcus aureus and Escherichia coli, inversely decreased. Moreover crosslinking provided prolonged controlled drug release with outstanding increase of mechanical properties observed by nanoindentation measurements. Results of the investigation indicated crosslinking as an important parameter for producing material with multifunctional characteristics suitable for drug delivery and tissue engineering.

Synergistic effect of divalent cations in improving technological properties of cross-linked alginate beads

Gelling solution parameters are some of the most important variables in ionotropic gelation and consequently influence the technological characteristics of the product. To date, only a few studies have focused on the simultaneous use of multiple cations as gelling agents. With the aim to deeply explore this possibility, in this research we investigated the effect of two divalent cations (Ca2+ and Zn2+) on alginate beads formation and properties. Alginate beads containing prednisolone (P) as model drug were prepared by prilling technique. The main critical variables of the ionotropic gelation process i.e. composition of the aqueous feed solutions (sodium alginate and prednisolone concentration) and cross-linking conditions (Ca2+, Zn2+ or Ca2++Zn2+), were studied. The obtained beads were characterized and their in vitro release performances were assessed in conditions simulating the gastrointestinal environment. Results evidenced a synergistic effect of the two cations, affecting positively both the encapsulation efficiency and the ability of the alginate polymeric matrix to control the drug release. A Ca2+/Zn2+ ratio of 4:1, in fact, exploited the Ca2+ ability of establish quicker electrostatic interactions with guluronic groups of alginate and the Zn2+ ability to establish covalent-like bonds with carboxylate groups of both guluronic and mannuronic moieties of alginate.

Optimization, characterization, and in vitro assessment of alginate-pectin ionic cross-linked hydrogel film for wound dressing applications

Natural polymer-based hydrogel films have great potential for biomedical applications and are good candidates for wound dressings. In this study, we aimed to develop simvastatin-loaded crosslinked alginate-pectin hydrogel films by ionic crosslinking to improve the mechanical characteristics, wound fluid uptake and drug release behavior. Alginate-pectin hydrocolloid films were chemically crosslinked by immersing in different concentrations of CaCl2 (0.5–3% w/v) for 2–20min. The degree of crosslinking was influenced by both contact time and CaCl2 concentration. The optimized conditions for crosslinking were 0.5% and 1% (CaCl2) for 2min. The optimized hydrogel films were then characterized for their physical, mechanical, morphological, thermal, in vitro drug release, and cytocompatibility profiles. Crosslinking improved the mechanical profile and wound fluid uptake capacity of dressings. The hydrogel films were able to maintain their physical integrity during use, and the best results were obtained with the film in which the extent of crosslinking was low (0.5%). Thermal analysis confirmed that the crosslinking process enhanced the thermal stability of hydrogel films. Sustained, slow release of simvastatin was obtained from the crosslinked films and in vitro cytotoxicity assay demonstrated that the hydrogel films were non-toxic.

A novel approach for fabricating highly tunable and fluffy bioinspired 3D poly(vinyl alcohol) (PVA) fiber scaffolds.

The excellent biocompatibility, biodegradability and chemo-thermal stability of poly(vinyl alcohol) (PVA) have been harnessed in diverse practical applications. These properties have motivated the fabrication of high performance PVA based nanofibers with adequate control over the micro and nano-architectures and surface chemical interactions. However, the high water solubility and hydrophilicity of the PVA polymer limits the application of the electrospun PVA nanofibers in aqueous environments owing to instantaneous dissolution. In this work, we report a novel yet facile concept for fabricating extremely light, fluffy, insoluble and stable three dimensional (3D) PVA fibrous scaffolds with/without coating for multifunctional purposes. While the solubility, morphology, fiber density and mechanical properties of nanofibers could be tuned by optimizing the cross-linking conditions, the surface chemical reactivity could be readily enhanced by coating with a polydopamine (pDA) bioinspired polymer without compromising the stability and innate properties of the native PVA fiber. The 3D pDA-PVA scaffolds exhibited super dye adsorption and constructive synergistic cell-material interactions by promoting healthy adhesion and viability of the human mesenchymal stem cells (hMSCs) within 3D micro-niches. We foresee the application of tunable PVA 3D as a highly adsorbent material and a scaffold material for tissue regeneration and drug delivery with close consideration of realistic in vivo parameters.

UV Dose Governs UV-Polymerized Polyacrylamide Hydrogel Modulus

Polyacrylamide (PAA) hydrogels have become a widely used tool whose easily tunable mechanical properties, biocompatibility, thermostability, and chemical inertness make them invaluable in many biological applications, such as cell mechanosensitivity studies. Currently, preparation of PAA gels involves mixtures of acrylamide, bisacrylamide, a source of free radicals, and a chemical stabilizer. This method, while generally well accepted, has its drawbacks: long polymerization times, unstable and toxic reagents, and tedious preparation. Alternatively, PAA gels could be made by free radical polymerization (FRP) using ultraviolet (UV) photopolymerization, a method which is quicker, less tedious, and less toxic. Here, we describe a simple strategy based on total UV energy for determining the optimal UV crosslinking conditions that lead to optimal hydrogel modulus.

Polyurethane Binder Mechanical Properties under Different Crosslinking Conditions

Mechanical properties of polyurethane type polymer binder with selected reactivity, used for production of the composite material based on scrap tires, were investigated under different crosslinking conditions: temperature, relative air humidity, hardening time. Shore C hardness, tensile strength and elongation at break were selected as main parameters, reflecting crosslinking degree of the prepared polymer binder films. DMA investigation of the samples in tensile mode of loading were realized to clear up influence of deformation circumstances on storage and loss components of the elasticity modulus. Strong correlation between mentioned above mechanical properties of the polymer binder and selected crosslinking conditions of the polymer were obtained. It was affirmed previously, that crosslinking degree of the polymer binder in the same time has direct influence also on mechanical properties of the composite material.

Strategy to Achieve Highly Porous/Biocompatible Macroscale Cell Blocks, Using a Collagen/Genipin-bioink and an Optimal 3D Printing Process.

Recently, a three-dimensional (3D) bioprinting process for obtaining a cell-laden structure has been widely applied because of its ability to fabricate biomimetic complex structures embedded with and without cells. To successfully obtain a cell-laden porous block, the cell-delivering vehicle, bioink, is one of the significant factors. Until now, various biocompatible hydrogels (synthetic and natural biopolymers) have been utilized in the cell-printing process, but a bioink satisfying both biocompatibility and print-ability requirements to achieve a porous structure with reasonable mechanical strength has not been issued. Here, we propose a printing strategy with optimal conditions including a safe cross-linking procedure for obtaining a 3D porous cell block composed of a biocompatible collagen-bioink and genipin, a cross-linking agent. To obtain the optimal processing conditions, we modified the 3D printing machine and selected an optimal cross-linking condition (∼1 mM and 1 h) of genipin solution. To show the feasibility of the process, 3D pore-interconnected cell-laden constructs were manufactured using osteoblast-like cells (MG63) and human adipose stem cells (hASCs). Under these processing conditions, a macroscale 3D collagen-based cell block of 21 × 21 × 12 mm3 and over 95% cell viability was obtained. In vitro biological testing of the cell-laden 3D porous structure showed that the embedded cells were sufficiently viable, and their proliferation was significantly higher; the cells also exhibited increased osteogenic activities compared to the conventional alginate-based bioink (control). The results indicated the fabrication process using the collagen-bioink would be an innovative platform to design highly biocompatible and mechanically stable cell blocks.

Insoluble poly(ethylene oxide) nanofibrous coating materials: effects of crosslinking conditions on the matrix stability

In this study, the effect of crosslinking conditions was investigated to obtain insoluble poly(ethylene oxide) (PEO) nanofiber mats having an ultraviolet (UV) initiating and crosslinking agent, pentaerythritol triacrylate (PETA), with various ratios in the presence or absence of UV irradiation at 366 nm. At first, PEO nanofibers were electrospun from 400,000 and 600,000 g/mole molecular weights of PEO and they were compared in terms of diameter and fiber morphology. Whereas applied voltage in the range of 10–25 kV had no significant effect on the fiber morphology, fiber diameters varied by voltage. An increase in the flow rate from 0.25 to 1.00 mL h−1 had an effect in favor of fabricating thicker fibers. The effect of distance to collector on the diameter and morphology was not distinctive. Fibers having irregular morphology and beads appeared with increasing the polymer concentration from 4 to 8 % w/v. Prior crosslinking, electrospinning process at selected conditions was applied to the PEO (600,000) including PETA and PEO-PETA nanofibers were obtained. Besides PETA concentration and UV application, drying conditions before UV irradiation were also found effective to obtain stable fibers in aqueous media. PEO nanofibers electrospun in the presence of 10 % PETA (w/w), dried for 8 days at 37 °C in an air atmosphere and then, irradiated with UV for 50 min were found most stable in aqueous media. However, crosslinking was also achieved in the absence of UV.

Comparison of glutaraldehyde and carbodiimides to crosslink tissue engineering scaffolds fabricated by decellularized porcine menisci

The objectives of this study were to fabricate porous scaffolds using decellularized meniscus, and to explore a preferable crosslinking condition to enhance mechanical properties of scaffolds. Moreover, the microstructure, porosity, biodegradation and cytotoxicity were also evaluated. EDAC or GTA in different concentration was used to crosslink scaffolds. FTIR demonstrated functional groups change in crosslinking process. SEM photography showed that crosslinked scaffolds had blurry edges, which resulted scaffolds crosslinked by 1.2mol/l EDAC had smaller porosity than other groups. The structure change enhanced antidegradation property. After immersing in enzyme solution for 96h, scaffolds crosslinked by GTA and EDAC could maintain their mass >70% and 80%. Most importantly, mechanical properties of crosslinked scaffolds were also improved. Uncrosslinked Scaffolds had only 0.49kPa in compression modulus and 12.81kPa in tensile modulus. The compression and tensile modulus of scaffolds crosslinked by 1.0% GTA were 1.42 and 567.44kPa respectively. The same value of scaffolds crosslinked by 1.2mol/l EDAC were 1.49 and 532.50kPa. Scaffolds crosslinked by 1.0% and 2.5% GTA were toxic to cells, while EDAC groups showed no cytotoxicity. Chondrocytes could proliferate and infiltrate within scaffolds after seeding. Overall, 1.2mol/l EDAC was a preferable crosslinking condition.

Preparation and characterization of cross-linked enzyme aggregates (CLEAs) of recombinant thermostable alkylsulfatase (SdsAP) from Pseudomonas sp. S9

Abstract SdsAP, an efficient SDS degradation alkylsulfatase from Pseudomonas sp. S9 , was immobilized in the form of cross-linked enzyme aggregates (CLEAs). Preliminary results revealed that over 80% activity of SdsAP-CLEAs was recovered using PEG4000 as the precipitating agent. Conditions for enzyme precipitation and cross-linking were further optimized. Compared to free SdsAP, SdsAP-CLEAs showed higher pH and temperature stability, and better tolerance to a certain organic solvents. Kinetic characterization analysis showed that SdsAP-CLEAs had higher affinity and catalytic efficiency than its soluble counterpart. Furthermore, SdsAP-CLEAs retained more than 60% of their initial activity after 10 batches of re-use at 50 °C and little or no loss of activity after one month at 4 °C. These results suggested that immobilization with CLEAs could improve the stability and operability of SdsAP, exhibiting a great potential application of SdsAP-CLEAs on SDS degradation in industry wastewater treatment.

Chitosan-based membranes with different ionic crosslinking density for pharmaceutical and industrial applications

Chitosan membranes (Ch), ionically crosslinked with pentasodium tripolyphosphate (TPP), were prepared using chitosan of medium and high molecular weight of similar degree of deacetylation and different crosslinking conditions. An effect of synthesis conditions (pH of crosslinking TPP solution equal to 5.5 and 9.0) on molecular and supermolecular structure and on crosslinking density of Ch/TPP membranes was confirmed using Fourier transform infrared spectroscopy (FTIR), wide-angle X-ray diffraction (WAXD) method and energy dispersive X-ray (EDX) spectrometry. Atomic-force microscopy (AFM) and contact angle measurements indicated some differences in membrane roughness and hydrophilicity. The state of water in non-crosslinked and ionically crosslinked Ch membranes containing different amount of water was investigated by low temperature differential scanning calorimetry (DSC) measurements. DSC analysis confirmed presence of freezing and non-freezing water in non-crosslinked and ionically crosslinked membranes. The amount of non-freezing water generally decreased after Ch crosslinking and was affected by crosslinking conditions and crosslinking density. Molecular weight of Ch had only slight influence on all characterized properties of ionically crosslinked membranes.