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Hydrogels have a wide range of applications in tissue engineering, drug delivery, device fabrication for biological studies and stretchable electronics. For biomedical applications, natural polymeric hydrogels have general advantages such as biodegradability and non-toxic by products as well as biocompatibility. However, applications of nature derived hydrogels have been severely limited by their poor mechanical properties. For example, most of the protein derived hydrogels do not exhibit high stretchability like methacrylated gelatin hydrogel has ∼11% failure strain when stretched. Moreover, protein derived elastomeric hydrogels that are fabricated from low molecular weight synthetic peptides require a laborious process of synthesis and purification. Biopolymers like gelatin, produced in bulk for pharma and the food industry can provide an alternative for the development of elastomeric hydrogels. Here, we report the synthesis of ureidopyrimidinone (Upy) functionalized gelatin and its fabrication into soft elastomeric hydrogels through supramolecular interactions that could exhibit high failure strain (318.73 ± 44.35%). The hydrogels were fabricated through a novel method involving co-solvent optimization and structural transformation with 70% water content. It is anticipated that the hydrogel fabrication method involves the formation of hydrophobic cores of ureidopyrimidinone groups inside the hydrogel which introduced elastomeric properties to the resulting hydrogel. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Nanyang Technological University Published version The authors acknowledge the financial support provided by Ministry of Education, Singapore, Singapore Centre for 3D Printing (SC3DP), A*STAR (Singapore Food Story R&D Programme), and School of Material Sciences and Engineering, Nanyang Technological University, Singapore.

Photopolymerization of protein-derived polymers functionalized with methacryloyl groups has been increasingly used to fabricate three-dimensional tissue constructs for biomedical applications because photocurable protein-based polymers (e.g., gelatin and collagen methacryloyl) feature spatial-temporal controllability of engineering complex constructs as well as inherent biological properties. Herein, we report photocurable albumin-based hydrogels. First, photocurable bovine serum albumin methacryloyl (BSA-MA) with different degrees of substitution (DM) was successfully synthesized in a precise manner, without substantially altering BSA native secondary structure. Resultant photocurable BSA-MA hydrogels exhibited tunable physio-biochemical properties over the swelling, degradation, and mechanical properties. Moreover, photo-cross-linked BSA-MA hydrogels provided a permissible environment to support cell viability and functionality both in two- and three-dimensional culture systems. Photocurable BSA-MA hydrogels may be used as a versatile platform for various bioapplications including tissue engineering and 3D bioprinting.

Hydrogels are three-dimensional (3D) polymeric networks that can confine a substantial fraction of water within their structure. Photopolymerization is one of the efficient methods to prepare hydrogels, by which polymers undergo a photocrosslinking process upon light exposure for a few minutes in the presence of an initiator. In comparison to other crosslinking methods (e.g. physical crosslinking and chemical addition/condensation crosslinking), photopolymerization has many advantages including fast and effective polymerization and good spatio-temporal control of hydrogel formation, and can occur under ambient reaction conditions at room temperature in physiological solutions. For these reasons, photocurable hydrogels have been extensively utilized for biomedical applications such as 3D hydrogel scaffold fabrication, drug delivery systems, and 3D bio-printing. This chapter begins with the introduction of photoinitiators, which play an important role in the preparation of photocurable hydrogels. Subsequently, two types of photocurable hydrogels and their preparation methods are described; the first section focuses on photocurable hydrogels made from synthetic materials, followed by the second section on those from natural materials.

This study was performed on two groups (10 cows) for primiparous Holstein lactating cows (av. 98 days in milk : DIM) which were divided into low forage diet (LF) and high forage diet (HF) groups based on forage : concentrate ratio (F : C ratio). The F : C ratios of LF and HF groups were 37:63 and 62:38, respectively. Concentrate intake was significantly higher in the LF group than the HF group whereas the HF group showed higher forage intake (12.9 kg) compared to the LF group (7.4 kg) (p 0.05), but the HF group tended to be higher. CP, TDN and NEL intake showed no significant difference between the two groups (p>0.05). Though, there was no significant difference on actual milk between the two groups (26 vs. 23.9 kg/d, p>0.05), the LF group tended to be higher. 4% FCMs of LF and HF groups were 22.8 and 22.3 kg/d, respectively, and showed no significant difference (p>0.05). The HF group was higher in fat content and lower in MUN. C14:0, C16:0 and C16:1n7 of milk fatty acid were significantly higher in the HF group (p

Gelatin-methacryloyl (GelMA) is one of the most commonly used photopolymerizable biomaterials in bio-applications. However, GelMA synthesis remains suboptimal, as its reaction parameters have not been fully investigated. The goal of this study is to establish an optimal route for effective and controllable GelMA synthesis by systematically examining reaction parameters including carbonate-bicarbonate (CB) buffer molarity, initial pH adjustment, MAA concentration, gelatin concentration, reaction temperature, and reaction time. We employed several analytical techniques in order to determine the degree of substitution (DS) and conducted detailed structural analysis of the synthesized polymer. The results enabled us to optimize GelMA synthesis, showing the optimal conditions to balance the deprotonation of amino groups with minimizing MAA hydrolysis, which led to nearly complete substitution. The optimized conditions (low feed ratio of MAA to gelatin (0.1 mL/g), 0.25 M CB buffer at pH 9, and a gelatin concentration of 10–20%) enable a simplified reaction scheme that produces GelMA with high substitution with just one-step addition of MAA in one pot. Looking forward, these optimal conditions not only enable facile one-pot GelMA synthesis but can also guide researchers to explore the efficient, high methacrylation of other biomacromolecules. NRF (Natl Research Foundation, S’pore) Published version

In this work, a comparison between two different physical-chemical gels, poly(NIPAAm-co-cysteamine) with poly(NIPAAm-co-cysteamine-vinylsulfone) and poly(NIPAAm-co-cysteamine) with poly(NIPAAm-co-HEMA-acrylate), is made. These hydrogels undergo gelation via dual mechanisms: temperature sensitivity (physical gelation) and chemical crosslinking (chemical gelation). The advantages of using both gelation mechanisms are to reduce the creep experienced by purely physical gels and to increase the elastic modulus of purely chemical gels. Here, the physical-chemical gels were synthesized and characterized for their chemical, structural, thermal, mechanical and morphological properties. The gels were also tested for their gelation kinetics, swelling, degradation and cytotoxicity. The copolymers were successfully synthesized and their phase transition temperatures fall in a feasible range (29-34°C) for use in vivo. With rheology, it was shown that use of simultaneous physical and chemical gelation resulted in improved properties, with increased elastic moduli and reduced frequency dependence. The rates of reaction of thiols to vinyls differ between the two systems, demonstrating a greater effect of chemical gelation in one gelling system over the other, due to the faster rate of thiols consumed into reaction. The morphology of the gels proved to be quite different when analyzed by scanning electron microscopy, showing differences in swelling behaviors. Cell studies illustrated good growth of cells exposed to the gels. Both hydrogels, although possessing slight differences, demonstrate the capability of being injected in vivo for use as embolic agents for occlusion of aneurysms.

A thermosensitive, injectable, and bioresorbable polymer hydrogel, poly(N-isopropylacrylamide-co-dimethyl-γ-butyrolactone acrylate-co-acrylic acid) [poly(NDBA)], was synthesized by radical copolymerization with 7.00 mol % dimethyl-γ-butyrolactone acrylate in tetrahydrofuran. The chemical composition was determined by acid titration in conjunction with (1) H NMR quantification. The molecular weight and polydispersity were determined by gel permeation chromatography in conjunction with static light scattering. The degradation properties of the polymer hydrogel were characterized using differential scanning calorimetry, percentage mass loss, cloud point test, and swelling ratio over time. It was found that the initial lower critical solution temperature (LCST) of the polymer is between room temperature and body temperature and that it takes about 2 weeks for the LCST to surpass body temperature under physiological conditions. An indirect cytotoxicity test indicated that this copolymer has relatively low cytotoxicity as seen with 3T3 fibroblast cells. The in vivo-gelation and degradation study showed good agreement with in vitro-degradation findings, and no detrimental effects to adjacent tissues were observed after the complete dissolution of the polymer. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.

Thermosensitive poly(organophosphazene) gels have been synthesized with a host of side groups, including α-amino-ω-methoxy-poly(ethylene glycol), hydrophobic amino acid esters (PheOEt, LeuOEt, and IleuOEt), depsipeptide ethyl ester (GlyGlycOEt), and lysine ethyl ester (lysOEt). The fraction of the last side group, lysOEt, which possesses two amine functional groups, was designed to be in the range of 0.1–0.3 mol per polymer unit. The poly(organophosphazenes) have been characterized via 1H- and 31P-NMR spectroscopies, GPC, and elemental analysis. The phase transition behavior of the poly(organophosphazenes) in aqueous solution has been determined via viscometry. Some of the poly(organophosphazenes) with amino functional groups exhibit reversible sol–gel transitions at temperatures near those of the human body, when in aqueous solution. These polymers form a sol at lower temperatures, and become gels at higher temperatures. Also, these polymer solutions have been found to behave generally like Newtonian fluids in the sol state, but appear to exhibit pseudoplastic qualities in the gel state. The polymers possessing depsipeptide ethyl esters (ethyl-2-(O-glycyl)glycolate) as a side group tend to exhibit much higher degradation rates under physiological conditions than do those which lack the depsipeptide ethyl ester group. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci 120:998–1005, 2011

BACKGROUND:In situ gelling polymers, like poly(N-isopropylacrylamide) (poly(NIPAAm)), have many potential medical applications due to their biocompatibility and thermosensitivity. RESULTS: Radio-opaque thermosensitive poly(NIPAAm) grafted with 10.7 wt% 2,2′-(ethylenedioxy)bis(ethylamine)-2,3,5-triiodobenzamide was successfully synthesized and characterized. The conjugated polymer showed good visibility with X-ray fluoroscopy. The polymer had a lower critical solution temperature of 30 °C after conjugation with triiodobenzamide as determined by cloud point determination and a transition peak temperature of 33.3 ± 0.57 °C as determined by differential scanning calorimetry. CONCLUSION: The polymer synthesized was highly visible under X-rays, based upon the percentage incorporation of triiodobenzamide. After conjugation of the NIPAAm to the triiodobenzamide through a bis(ethylamine) linkage, the resultant polymer retained lower critical solution temperature characteristics in a temperature region that makes it physiologically useful. Copyright © 2009 Society of Chemical Industry

Novel, bioerodible, thermosensitive poly(NIPAAm-co-dimethyl–γ-butyrolactone), with hydrolysis-dependent thermosensitivity, were synthesized by radical polymerization with varying dimethyl-γ-butyrolactone content and the properties of the copolymers were characterized using Differential Scanning Calorimetry (DSC), High Performance Liquid Chromatography (HPLC) in conjunction with Static Light Scattering, Fourier Transformed Infrared Spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR) and acid titration. The lower critical solution temperature (LCST) of the copolymers decreased with increasing dimethyl-γ-butyrolactone content, but then increased after ring-opening hydrolysis of the dimethyl-γ-butyrolactone side group. FTIR and NMR spectra showed the copolymerization of these two monomers and the hydrolysis-dependent ring-opening of the dimethyl-γ-butyrolactone side group. It was also found that there are no low-molecular-weight by-products but rather dissolution of the polymer chains at 37°C during the time frame of application. Models of the kinetics suggest that the hydrolysis reaction is self-catalytic due to an increase in hydrophilicity, and thus accessible water concentration, caused by ring-opening of the dimethyl-γ-butyrolactone.

In an effort to create an in situ physically and chemically cross-linked hydrogel for in vivo applications, N-isopropylacrylamide (NIPAAm) was copolymerized with poly(ethylene glycol)-monoacrylate (PEG-monoacrylate) and then the hydroxyl terminus of the PEG was further modified with acryloyl chloride to form poly(NIPAAm-co-PEG) with acrylate terminated pendant groups. In addition to physically gelling with temperature changes, when mixed with a multi-thiol compound such as pentaerythritol tetrakis 3-mercaptopropionate (QT) in phosphate buffer saline solution of pH 7.4, this polymer formed a chemical gel via a Michael-type addition reaction. The chemical gelation time of the polymer was affected by mixing time; swelling of the copolymer solutions was temperature dependant. Because of its unique gelation properties, this material may be better suited for long-term functional replacement applications than other thermo-sensitive physical gels. Also, the PEG content of this material may render it more biocompatible than similar HEMA-based precursors in previous simultaneous chemically and physically gelling materials. With its improved mechanical strength and biocompatibility, this material could potentially be applied as a thermally gelling injectable biomaterial for aneurysm or arteriovenous malformation (AVM) occlusion. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007

Thermosensitive poly(organophosphazenes) bearing α-amino-ω-methyl-poly(ethylene glycol) (AMPEG), some hydrophobic amino acid esters, and a depsipeptide ethyl ester (ethyl-2-(O-glycyl)glycolate) as a hydrolysis-sensitive moiety have been synthesized. Most of the poly(organophosphazenes) synthesized showed sol−gel transition properties in an aqueous solution. The gelation properties of the polymers were dependent on several factors like the composition of substituents, the chain length of AMPEG, and kinds of amino acid esters. The gelation of the polymers seemed to be imparted by association of hydrophobic groups of amino acid esters. The polymers decomposed under the physiological conditions. The polymer with a small amount of depsipeptide was hydrolyzed more rapidly than that with no depsipeptide. The viscosity of the polymer gels was also affected by their degradation rate.

higher content of MPEG and shorter chain length of alkylamines of the polymers afforded the higher LCST. The LCSTs of the polymers exhibited almost concentration-independent behavior in the range of 3-30 wt % of the polymers in aqueous solutions. The polymers showed the higher LCSTs in the acidic solutions than in the neutral and basic solutions. The ionic strength of the polymer solution affected the LCST, which decreased with increased NaCl concentration. The polymer bearing almost equimolar substitutuents with the -N-P-N- unit has shown the LCST more sensitive to NaCl and pH than that with the -N-P-O- unit. The polymers were found to degrade in acidic solution but be very stable in alkali solution as well as in the buffer solution of pH 7.4.

The lower critical solution temperature (LCST) properties and hydrolytic behaviour of thermosensitive biodegradable polyphosphazenes with monomethoxy-poly(ethylene glycol), glycine ethyl ester and depsipeptide ethyl esters substituents have been studied. All the polymers synthesized show LCST properties. The LCSTs of the polymers are affected by the composition of the polymers and increase with the degradation of the polymers in aqueous solution. The higher content of depsipeptide ethyl esters in the polymers accelerates the LCST change and the degradation of the polymers in aqueous solution. Most polymers synthesized have half-lives of less than 10 days in neutral solution. © 2002 Society of Chemical Industry

The aim of this work was to develop a simultaneous physically and chemically gelling system using NIPAAm co-polymers. The in situ polymer gel was obtained by synthesizing poly(NIPAAm-co-HEMAacrylate) and poly(NIPAAm-co-cysteamine) through free radical polymerization and further nucleophilic substitution. The purpose of the dual gelation is that physical gelation would take place at higher temperatures as the NIPAAm chains associate, while chemical gelation would occur through a Michael-type addition reaction, resulting in a cross-link forming through a nucleophilic attack of the thiolate on the acrylate. The structure of each co-polymer was then verified using (1)H-NMR and FT-IR spectroscopy. The corresponding lower critical solution temperature and phase transition behavior of each co-polymer was analyzed through cloud point and DSC, while mechanical properties were investigated through rheology. Swelling behavior was also monitored at different temperatures. The resulting polymer system demonstrated properties compatible with physiological conditions, forming a gel at pH 7.4 and at temperatures near body temperature. The hydrogel also showed reduced viscoelastic flow at low frequency stress, and increased strength than purely physical or chemical gels. Swelling behavior was determined to be temperature-dependent; however, no difference was observed in swelling percent beyond 48 h. Having the ability to alter these co-polymers through various synthesis parameters and techniques, this hydrogel can potentially be used as an injectable, waterborne gelling material for biomedical applications such as endovascular embolization.

X-ray visibility is an integral design component of in situ gelling embolization systems for neurovascular treatment. The goals of this project included the synthesis and characterization of a unique intrinsically radio-opaque in situ gelling material for neurovascular embolization. The gels formed using Michael-Type Addition between pentaerythritol tetrakis 3-mercaptopropionate (QT) thiols and poly(propylene glycol) diacrylate (PPODA) with the addition of the new material Iodobenzoyl poly(ethylene glycol) acrylate (IPEGA), a radio-opaque agent, synthesized successfully as confirmed with (1)H NMR. The PPODA and IPEGA were mixed using a syringe coupler with QT and buffer at pH 11 for 90 seconds. Gel mixes were weighed to provide equal molar thiols and acrylate groups, changing the present acrylate-bearing compounds wt % ratios from 100 PPODA: 0 IPEGA, 90:10, 80:20, 70:30, 60:40, 50:50, and 0:100. Formulations with 10% and above of IPEGA were X-ray visible. Rheology showed that increasing the amount of IPEGA decreased the storage. Kinetic FT-IR studies indicate that the amphiphilic nature of the PEG backbone increased the reaction rate of the phase segregated reactants. Second order reaction constant modeling showed a change in initial reaction rate from 0.0029 to 0.0187 (M sec)(-1) from the 10% to 50% IPEGA formulations respectively.

To ensure patients' safety during vascular surgery involving polymer injection, it is important to ensure a high degree of control over the gelation rate of the materials to prevent the formation of uncontrolled emboli from shear or dispersion. Through manipulation of factors affecting the kinetics of in situ forming polymeric systems, patients' safety can be maximized. The rate of Michael-Type Addition is known to be affected by the pH of the accompanying solution, as thiols will become nucleophilic thiolates in an aqueous environment. This work evaluates the effect of the addition of 2 and 4 wt % of several common surfactants on the gelation rate of reverse emulsion polymeric systems as a method for augmenting control of in vivo Michael-type addition kinetics. This work shows that both Tween-20 (HLB 16.7) and Pluronic type surfactants (HLB reverse similar 6.7) stabilize our emulsion, with gelation rates being significantly different in the presence of Pluronic type surfactants (p < 0.05). This work has demonstrated the ability of surfactants to promote faster gelation at lower pH by maximizing emulsion stability and reducing cohesion of smaller bubbles, thereby maximizing surface area between the initiator and the bulk phases.

This work investigates the differential reaction rates seen among several Michael-Type acceptors when reacted with poly(NIPAAm-co-cysteamine). This work differs from many of the previous studies upon mercaptans in that it examines systems used for network and gel formation. We find that the reaction rates of poly(NIPAAm-co-cysteamine) cross-linked with Michael type acceptors follow traditional second order rate laws. In addition, we further confirm that these reactions are pH sensitive, reliant upon the pK (a) of the conjugated thiols, and on local chain chemistry. Additionally, this work determines that the reaction of difunctional acrylates with the macromolecular NIPAAm molecules leads to an apparent, but not significant, increase in the rate of reaction. The low magnitude of this increase is likely indicative of increased steric hindrance arising from network formation, or reduced diffusion in the NIPAAm polymer chains. Statistical analysis shows pH and ratio of thiol to acrylates significantly affect reaction rates (p0.05). The type of acrylate (PEGDA, PEGMA, or HEA) does not return as significant globally or within a pH range. Since localizing charge on a chain raises the effective pK (a) of nearby acids, gains in reaction rate from increasing chain functionality are shown to increase much less than would be expected from the increased concentration.

The objective of this work is to create an in situ physically and chemically cross-linking hydrogel for in vivo applications. N-isopropylacrylamide (NIPAAm) was copolymerized with N-acryloxysuccinimide (NASI) via free radical polymerization. Poly(NIPAAm-co-NASI) was further modified to obtain Poly(NIPAAm-co-cysteamine) through a nucleophilic attack on the carbonyl group of the NASI by the amine group of the cysteamine. Modification was verified by nuclear magnetic resonance. In addition to thermo-responsive physical gelling due to the presence of NIPAAm, this system also chemically gels via a Michael-Type addition reaction when mixed with poly(ethylene glycol) diacrylate (PEGDA). The presence of both physical and chemical gelation resulted in material properties that are much improved compared to purely physical gels. The chemical gelation time of the copolymers was not significantly affected by the amount of thiol present due to the increased pKa of the copolymer containing more thiols. In addition, the swelling of the copolymers was highly dependent on temperature and thiol content. Lastly, the rate of nucleophilic attack in the Michael-Type addition reaction was shown to be highly dependent on pH and on the mole ratio of thiol to acrylate. Due to the improved mechanical properties, this material may be better suited for long-term functional replacement applications than other thermo-sensitive physical gels. With further development and biocompatibility testing, this material could potentially be applied as a temperature-responsive injectable biomaterial for functional embolization.

The goal of this work is to make an injectable physically and chemically cross-linking NIPAAm-based copolymer system for endovascular embolization. A copolymer with N-isopropylacrylamide (NIPAAm) and hydroxyethyl methacrylate (HEMA) was synthesized and converted to poly(NIPAAm-co-HEMA-acrylate) functionalized with olefins. When poly(NIPAAm-co-HEMA-acrylate) was mixed with pentaerythritol tetrakis 3-mercaptopropionate (QT) stoichiometrically in a 0.1 N PBS solution of pH 7.4, it formed a temperature-sensitive hydrogel with low swelling through the Michael-type addition reaction and showed improved elastic properties at low frequency compared to physical gelation. This material could be useful for applications requiring water-soluble injection but lower swelling and lower creep properties than available with other soluble in-situ-gelling materials.

Copolymers of N-isopropylacrylamide, 2-hydroxyethyl methacryl lactate [(HEMA)-lactate] and acrylic acid (AAc) were prepared with varying mole ratios of monomers to develop copolymers with gelation properties above a certain concentration for a bioerodible, in-situ gelling material. The copolymers formed gels in situ under physiological condition. The gelation temperature of the copolymers decreased as the HEMA-lactate content of the copolymers increased due to the hydrophobicity of HEMA-lactate, and increased as the AAc content increased due to the hydrophilicity of AAc. The gels redissolve at 37 degrees C as their LCSTs increase above 37 degrees C due to the hydrolysis of the HEMA-lactate pendant groups.

To develop a new class of in situ-forming, injectable, and biodegradable polymeric biomaterials based on time-dependent lower critical solution temperature (LCST) properties for localized delivery, copolymers of N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacryl lactate (HEMA-lactate) and acrylic acid (AAc) were prepared with varying mole ratios of monomers. The copolymers showed LCST and gelation properties below body temperature in 0.1 N PBS solution of pH 7.4. The LCST and gelation temperature of the copolymers decreased as the HEMA-lactate content of the copolymers was increased. The copolymers also showed time-dependent LCST and gelation properties in 0.1 N PBS solution of pH 7.4 owing to hydrolysis of HEMA-lactate. Hydrolysis of HEMA-lactate caused the polymers to be more hydrophilic, resulting in an increase in LCST and gelation temperature. All the polymers with about 6 mol % AAc exhibited LCST and gelation temperature above body temperature after complete hydrolysis of HEMA-lactate.

Considerable interest has arisen in precision fabrication of cell bearing scaffolds and structures by free form fabrication. Gelatin is an ideal material for creating cell entrapping constructs, yet its application in free form fabrication remains challenging. We demonstrate the use of gelatin, crosslinked with microbial transglutaminase (mTgase), as a material to print cell bearing hydrogels for both 2-dimensional (2-D) precision patterns and 3-dimensional (3-D) constructs. The precision patterning was attained with 3 % gelatin and 2 % high molecular weight poly (ethylene oxide) (PEO) whereas 3-D constructs were obtained using a 5 % gelatin solution. These hydrogels, referred to as “bioinks” supported entrapped cell growth, allowing cell spreading and proliferation for both HEK293 cells and Human Umbilical Vein Endothelial Cells (HUVECs). These bioinks were shown to be dispensable by robotic precision, forming patterns and constructs that were insoluble and of suitable stiffness to endure post gelation handling. The two bioinks were further characterized for fabrication parameters and mechanical properties. Electronic supplementary material The online version of this article (doi:10.1007/s10544-014-9915-8) contains supplementary material, which is available to authorized users.