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Immunocompatibility and non-thrombogenicity are important requirements for biomedical applications such as vascular grafts. Here, gelatin-based hydrogels formed by reaction of porcine gelatin with increasing amounts of lysine diisocyanate ethyl ester were investigated in vitro in this regard. In addition, potential adverse effects of the hydrogels were determined using the “Hen’s egg test on chorioallantoic membrane” (HET-CAM) test and a mouse model. The study revealed that the hydrogels were immunocompatible, since complement activation was absent and a substantial induction of reactive oxygen species generating monocytes and neutrophils could not be observed in whole human blood. The density as well as the activation state of adherent thrombocytes was comparable to medical grade polydimethylsiloxane, which was used as reference material. The HET-CAM test confirmed the compatibility of the hydrogels with vessel functionality since no bleedings, thrombotic events, or vessel destructions were observed. Only for the samples synthesized with the highest LDI amount the number of growing blood vessels in the CAM was comparable to controls and significantly higher than for the softer materials. Implantation into mice showed the absence of adverse or toxic effects in spleen, liver, or kidney, and only a mild lymphocytic activation in the form of a follicular hyperplasia in draining lymph nodes (slightly increased after the implantation of the material prepared with the lowest LDI content). These results imply that candidate materials prepared with mid to high amounts of LDI are suitable for the coating of the blood contacting surface of cardiovascular implants.

The establishment of confluent endothelial cell (EC) monolayers on implanted materials has been identified as a concept to avoid thrombus formation but is a continuous challenge in cardiovascular device engineering. Here, material properties of gelatin-based hydrogels obtained by reacting gelatin with varying amounts of lysine diisocyanate ethyl ester were correlated with the functional state of hydrogel contacting venous EC (HUVEC) and HUVEC's ability to form a monolayer on these hydrogels. The density of adherent HUVEC on the softest hydrogel at 37 °C (G' = 1.02 kPa, E = 1.1 ± 0.3 kPa) was significantly lower (125 mm-1) than on the stiffer hydrogels (920 mm-1; G' = 2.515 and 5.02 kPa, E = 4.8 ± 0.8 and 10.3 ± 1.2 kPa). This was accompanied by increased matrix metalloprotease activity (9 pmol·min-2 compared to 0.6 pmol·min-2) and stress fiber formation, while cell-to-cell contacts were comparable. Likewise, release of eicosanoids (e.g., prostacyclin release of 1.7 vs 0.2 pg·mL-1·cell-1) and the pro-inflammatory cytokine MCP-1 (8 vs

Hydrophilic biopolymers display a strong tendency for self-organization into stable secondary, tertiary, and quaternary structures in aqueous environments. These structures are sensitive to changes in external conditions, such as temperature, pH or ions/salts, which may lead to molecular and/or macroscopic transitions. Here, we report on biopolymer-based stimuli-sensitive switchable matrices showing a shape-memory function as an output being alternatively switched by two different input signals, such as environmental changes in salt concentration or temperature. This was realized by implementing a shape-memory function in hydrogels based on the coil-to-helix transition of protein chains in gelatin-based networks. The hydrogels exhibited mechanical properties similar to that of soft tissue (storage modulus G′ = 1–100 kPa) and high swelling capabilities (Q = 1000–3000 vol %). In these gelatin-based networks, the covalent netpoints defined the permanent shape while after deformation helicalization of the gelatin acted as reversible stimuli-sensitive switches providing additional crosslinks capable of fixing the deformed temporary shape. By using either chaotropic salts to suppress gelatin helicalization or kosmotropic salts to support conformational changes of gelatin toward a helical orientation, these additional crosslinks could be cleaved or formed. In bending experiments, the strain fixity (Rf) and strain recovery ratios (Rr) were determined. While Rf ranged from 65 to 95% and was depending on the network composition, Rr were independent of the hydrogel composition with values about 100%. In addition, Rf and Rr were independent of the type of chaotropic salt that was used in this study, showing equal Rf and Rr values for MgCl2, NaSCN, and Mg(SCN)2.

Hydrogel forming physical networks based on gelatin are an attractive approach toward multifunctional biomaterials with the option of reshaping, self-healing, and stimuli-sensitivity. However, it is challenging to design such gelatin-based hydrogels to be stable at body temperature. Here, gelatin functionalized with desaminotyrosine (DAT) or desaminotyrosyl tyrosine (DATT) side chains is crosslinked with cyclodextrin (CD) dimers under formation of inclusions complexes. The supramolecular networks displayed at room temperature decreased water uptake (200-600 wt% for DAT-based systems, 200 wt% for DATT based systems), and increased storage moduli up to 25.6 kPa determined by rheology compared to DAT(T) gelatin. The gel-sol transition temperature increased from 33 up to 42 °C. The presented system that is completely based on natural building blocks may form the basis for materials that may potentially respond by dissolution or changes of properties to changes in environmental conditions or to the presence of CD guest molecules.

While click chemistry reactions for biopolymer network formation are attractive as the defined reactions may allow good control of the network formation and enable subsequent functionalization, tailoring of gelatin network properties over a wide range of mechanical properties has yet to be shown. Here, it is demonstrated that copper-catalyzed alkyne-azide cycloaddition of alkyne functionalized gelatin with diazides gave hydrogel networks with properties tailorable by the ratio of diazide to gelatin and diazide rigidity. 4,4′-diazido-2,2′-stilbenedisulfonic acid, which has been used as rigid crosslinker, yielded hydrogels with Young’s moduli E of 50–390 kPa and swelling degrees Q of 150–250 vol.%, while the more flexible 1,8-diazidooctane resulted in hydrogels with E = 125–280 kPa and Q = 225–470 vol.%. Storage moduli could be varied by two orders of magnitude (G′ = 100–20,000 Pa). An indirect cytotoxicity test did not show cytotoxic properties. Even when employing 1:1 ratios of alkyne and azide moieties, the hydrogels were shown to contain both, unreacted alkyne groups on the gelatin backbone as well as dangling chains carrying azide groups as shown by reaction with functionalized fluorescein. The free groups, which can be tailored by the employed ratio of the reactants, are accessible for covalent attachment of drugs, as was demonstrated by functionalization with dexamethasone. The sequential network formation and functionalization with click chemistry allows access to multifunctional materials relevant for medical applications.

Polymeric matrices mimicking multiple functions of the ECM are expected to enable a material induced regeneration of tissues. Here, we investigated the adipogenic differentiation of human adipose derived mesenchymal stem cells (hADSCs) in a 3D architectured gelatin based hydrogel (ArcGel) prepared from gelatin and L-lysine diisocyanate ethyl ester (LDI) in an one-step process, in which the formation of an open porous morphology and the chemical network formation were integrated. The ArcGel was designed to support adipose tissue regeneration with its 3D porous structure, high cell biocompatibility, and mechanical properties compatible with human subcutaneous adipose tissue. The ArcGel could support initial cell adhesion and survival of hADSCs. Under static culture condition, the cells could migrate into the inner part of the scaffold with a depth of 840 +/- 120 mu m after 4 days, and distributed in the whole scaffold (2mm in thickness) within 14 days. The cells proliferated in the scaffold and the fold increase of cell number after 7 days of culture was 2.55 +/- 0.08. The apoptotic rate of hADSCs in the scaffold was similar to that of cells maintained on tissue culture plates. When cultured in adipogenic induction medium, the hADSCs in the scaffold differentiated into adipocytes with a high efficiency (93 +/- 1%). Conclusively, this gelatin based 3D scaffold presented high cell compatibility for hADSC cultivation and differentiation, which could serve as a potential implant material in clinical applications for adipose tissue reparation and regeneration.

Cell-based therapies often face the challenge of low cell retention and viability upon transplantation. Hence, biomaterials, which can immobilize transplanted cells, while at the same time support cell viability, are essential for successful clinical application. Noteworthy, biomaterials in the micrometer range such as microcapsules or microspheres have the advantage of a minimally invasive introduction into tissue.Hence, we established an approach to generate gelatin-based cell carriers in the form of microspherical hydrogels. Fibroblasts were microencapsulated in glycidylmethacrylate (GMA)-functionalized gelatin by photopolymerization. While the degree of GMA-functionalization was kept constant, the hydrogel cross-linking density was adjusted by varying the time of irradiation or the average gelatin-chain length.Stable microspheres were synthesized from 10 wt% GMA-gelatin solutions for all irradiation periods tested (0.5 -2 min). Evaluation of cell viability revealed that microgels with the same weight content of biopolymer but with decreased cross-linking densities and thus decreased storage and E modulus, resulted in best cell support. Noteworthy, encapsulated cells partially migrated out of the microspheres and attached to the spherical surface.10 wt% GMA-gelatin-based hydrogels with E moduli comparable to the native cellular niche proved to be a promising biomaterial suitable for the production of cell-laden microspheres and shall be evaluated further for biomedical application.

Angiogenesis is one of the key processes during development, wound healing and tumor formation. Prerequisite for its existence is the presence of endogenous electrical fields (EFs) generated by active ion transport across polarized epithelia and endothelia, and appearance of the transcellular potentials. During angiogenesis cellular factor as endothelial growth factor (VEGF), synthesis of adhesive proteins and membrane metalloproteinases (MMPs) govern the angiogenic response to different external stimuli as biomaterials interactions and/or exogenous EF. Gelatin-based hydrogels with elasticities comparable to human tissues have shown to influence cell behavior as well as cell attachment, protein synthesis, VEGF and MMP's production after the application of EF. Gelatin-based matrices with 3 (G10_LNCO3), 5 (G10_LNCO5), and 8 (G10_LNCO8) fold excess of isocyanate groups per mol of amine groups present in gelatin were used. Human umbilical endothelial cells (HUVEC) (Lonza Basel, Switzerland) and highly invasive breast cancer MDA-MB-231 cells (ATCC®HTB-26TM) were used. For an estimation of the amount of VEGF released from cells a commercially available VEGF ELISA (Thermo Fisher Scientific, Germany) kit was used. Fibronectin (FN) enzyme immunoassay (EIA) was used to analyze the secreted amount of FN by cells seeded on the materials. Secreted MMPs were analyzed by zymography. Gelatin-based hydrogels attracted HUVEC adhesion and diminished the adhesion of MDA-MB-231 cells. The applied direct current (DC) EF induced an almost 5-fold increase in VEGF production by HUVEC seeded on gelatin-based hydrogels, while in contrast, the applied EF decreased the production of VEGF by cancer cells. FN synthesis was elevated in HUVEC cells seeded on gelatin-based materials in comparison to FN synthesis by cancer cells. HUVEC seeded on gelatin hydrogels showed an expression mainly of MMP-2. The application of EF increased the production of MMP-2 in HUVEC seeded on gelatin materials. In contrast, for MDA-MB-231 the production of MMPs on gelatin materials was lower compared to control materials. With the application of EF the levels of MMP-9 decreased but MMP-2 expression raised significantly for gelatin materials. Overall, the results showed that studied gelatin materials suppressed attachment of cancerous cells, as well as suppressed their angiogenic potential revealed by decreased VEGF and MMP production. Thus, this study approved gelatin-based hydrogels with proper elasticity characteristics and different degradation behavior as useful matrices for use in vascular tissue regeneration or in restriction of tumor growth after tumor resection.

Encapsulation by polymeric biomaterials can provide mechanical protection of cells and shielding from the immune system of the host when implanted as cell therapy. At the same time, free exchange of nutrients and metabolites including bioactive molecules guiding regenerative processes is facilitated. Here, glycidylmethacrylated gelatin (GMA-gelatin) is explored as matrix material for adherent (L929 mouse fibroblasts) or non-adherent (Ramos blue) cells by an integrated process of shaping and chemical crosslinking. Microparticle formation was driven by a water-in-water-emulsion technique, which allowed simultaneous irradiation with light of 365 nm in the presence of the photosensitizer irgacure 2959. Suitable photopolymerization conditions were determined in experiments with GMA-gelatin and cells. More than 85% of the cells survived this procedure, and an encapsulation efficiency of up to 75 ± 2% was reached. Diffusivity of molecules up to a molar mass of 150 kg·mol−1 in the matrix was shown by the release of co-encapsulated FITC-labelled dextran. L929 as well as Ramos blue cells proliferated in the microparticle matrix after encapsulation and released enzymes that could be detected in the cell culture medium in an active form. L929 showed the ability to escape the particles over time. Altogether, the presented cell encapsulation system based on a material that is stable to hydrolytic degradation for several weeks is generally suitable for cell based therapy or in vitro test systems.

Polymer degradation occurs under physiological conditions in vitro and in vivo, especially when bonds susceptible to hydrolysis are present in the polymer. Understanding of the degradation mechanism, changes of material properties over time, and overall rate of degradation is a necessary prerequisite for the knowledge-based design of polymers with applications in biomedicine. Here, hydrolytic degradation studies of gelatin-based networks synthesized by copper-catalyzed azide-alkyne cycloaddition reaction are reported, which were performed with or without addition of an enzyme. In all cases, networks with a stilbene as crosslinker proofed to be more resistant to degradation than when an octyl diazide was used. Without addition of an enzyme, the rate of degradation was ruled by the crosslinking density of the network and proceeded via a bulk degradation mechanism. Addition of Clostridium histolyticum collagenase resulted in a much enhanced rate of degradation, which furthermore occurred via surface erosion. The mesh size of the hydrogels (>7 nm) was in all cases larger than the hydrodynamic radius of the enzyme (4.5 nm) so that even in very hydrophilic networks with large mesh size enzymes may be used to induce a fast surface degradation mechanism. This observation is of general interest when designing hydrogels to be applied in the presence of enzymes, as the degradation mechanism and material performance are closely interlinked. Copyright © 2016 John Wiley & Sons, Ltd.

Gelatin-based hydrogels offer various biochemical cues that support encapsulated cells and are therefore suitable as cell delivery vehicles in regenerative medicine. However, besides the biochemical signals, biomechanical cues are crucial to ensure an optimal support of encapsulated cells. Hence, we aimed to correlate the cellular response of encapsulated cells to macroscopic and microscopic elastic properties of glycidylmethacrylate (GMA)-functionalized gelatin-based hydrogels. To ensure that different observations in cellular behavior could be attributed to differences in elastic properties, an identical concentration as well as degree of functionalization of biopolymers was utilized to form covalently crosslinked hydrogels. Elastic properties were merely altered by varying the average gelatin-chain length. Hydrogels exhibited an increased degree of swelling and a decreased bulk elastic modulus G′ with prolonged autoclaving of the starting solution. This was accompanied by an increase of hydrogel mesh size and thus by a reduction of crosslinking density. Tougher hydrogels retained the largest amount of cells; however, they also interfered with cell viability. Softer gels contained a lower cell density, but supported cell elongation and viability. Observed differences could be partially attributed to differences in bulk properties, as high crosslinking densities interfere with diffusion and cell spreading and thus can impede cell viability. Interestingly, a microscopic elastic modulus in the range of native soft tissue supported cell viability and elongation best while ensuring a good cell entrapment. In conclusion, gelatin-based hydrogels providing a soft tissue-like microenvironment represent adequate cell delivery vehicles for tissue engineering approaches. Copyright © 2016 John Wiley & Sons, Ltd.

Functionalization of gelatin with desaminotyrosine (DAT) and desamino tyrosyl tyrosine (DATT) has been demonstrated to allow network formation based on non-covalent interactions of the aromatic moieties. Based on the observation that the DAT(T) groups furthermore could interact with hydroxyapatite fillers, here it was investigated whether such interactions of DAT(T) could also be employed to stabilize composites formed by functionalized gelatins and bioactive glass (BG) particles. Because of sedimentation of the BG microparticles during the gelification, anisotropic composites with two distinct layers were formed. The characterization of mechanical properties by tensile tests and rheology showed that all composites of non-functionalized and DAT(T) functionalized gelatins with BG microparticles showed an increased Young’s modulus (E) up to 3 MPa, an increased storage modulus (G’) up to 100 kPa, increased tensile strength (σmax) up to 3.4 MPa, and increased loss modulus (G’’) compared to the pure matrices. As the observed effects were more pronounced in the DAT(T) functionalized gelatins compared to non-functionalized gelatins, and a much increased thermal stability of these composites was found, it is likely that there are binding interactions between the aromatic moieties and the BG microparticles. This effect open opportunities for the further development of this type of gelatin-based composites for bone regeneration applications.

Gelatin functionalized with glycidyl methacrylate (GMA) has been shown to allow crosslinking by photopolymerization and metathesis reaction. However, side chain functionalization of gelatin might reduce triple helicalization, which influences mechanical properties of gelatin-based polymer networks. Here, the influence of glycidylmethycrylation of gelatin on the chain organization, swelling, and mechanical properties is investigated by comparing among each other physical gels prepared from GMA-gelatin solutions of different concentrations (5-20 wt.-%) by drying and rehydration. An increase of GMA-gelatin concentration from 5 wt.-% to 20 wt.-% led to an increased density of produced gelatin films and a decreasing water uptake of the films from 1160 wt.-% to 730 wt.-%, while the storage modulus was increasing about one order of magnitude from 440 Pa to 4090 Pa. The relative single and triple helix content was not influenced by the variation of polymer concentration.

Hydrogels prepared from gelatin and lysine diisocyanate ethyl ester provide tailorable elastic properties and degradation behavior. Their interaction with human aortic endothelial cells (HAEC) as well as human macrophages (Mɸ) and granulocytes (Gɸ) were explored. The experiments revealed a good biocompatibility, appropriate cell adhesion, and cell infiltration. Direct contact to hydrogels, but not contact to hydrolytic or enzymatic hydrogel degradation products, resulted in enhanced cyclooxygenase-2 (COX-2) expression in all cell types, indicating a weak inflammatory activation in vitro. Only Mɸ altered their cytokine secretion profile after direct hydrogel contact, indicating a comparably pronounced inflammatory activation. On the other hand, in HAEC the expression of tight junction proteins, as well as cytokine and matrix metalloproteinase secretion were not influenced by the hydrogels, suggesting a maintained endothelial cell function. This was in line with the finding that in HAEC increased thrombomodulin synthesis but no thrombomodulin membrane shedding occurred. First in vivo data obtained after subcutaneous implantation of the materials in immunocompetent mice revealed good integration of implants in the surrounding tissue, no progredient fibrous capsule formation, and no inflammatory tissue reaction in vivo. Overall, the study demonstrates the potential of gelatin-based hydrogels for temporal replacement and functional regeneration of damaged soft tissue.

Ring-opening metathesis catalysis has received little attention as a means to functionalize or crosslink biopolymers in water since the required catalysts are usually not stable under these conditions. However, biopolymer solubility suggests such a procedure. We show that Grubbs first and second generation catalysts (in emulsion) as well as a water-soluble Hoveyda-Grubbs catalyst can be applied to crosslink glycidyl methacrylated gelatin with norbornene dicarboxylic acid in water by a cross metathesis-ring opening metathesis approach. A mechanistic study suggests that cross metathesis between the functionalized polymer and the cyclic olefin acts as an initiating step for the crosslinking reaction. Copyright © 2014 John Wiley & Sons, Ltd.

Summary Side chain functionalization of gelatin with tyrosine-derived moieties, desaminotyrosine (DAT) or desaminotyrosyl tyrosine (DATT), has been reported to lead to physical networks stabilized by aromatic interactions and hydrogen bonds, while the inherent ability of gelatin chains to organize in helices is suppressed. Here, the treatment of DAT and DATT gelatin films at defined temperatures (drying at 5 °C, freeze-drying, and freeze-thawing) were explored for the potential to additionally stabilize the hydrogels by increasing the content of helical domains as additional physical netpoints. The influence of the drying procedures on the hydrogel properties such as network morphology and mechanical properties were analyzed by WAXS, swelling, and rheological measurements. The triple helix content had a stabilizing effect on gelatin-based hydrogels at temperatures below the helix-to-coil transition. However, this effect was less pronounced at physiological conditions above the transition temperature, resulting in rapid dissolution of the physical gelatin networks.

Polymer network formation is an important tool for tailoring mechanical properties of polymeric materials. One option to synthesize a network is the addition of bivalent crosslinkers reacting with functional groups present in a polymer. In case of polymer network syntheses based on biopolymers, performing such a crosslinking reaction in water is sometimes necessary in view of the solubility of the biopolymer, such as gelatin, and can be beneficial to avoid potential contamination of the formed material with organic solvents in view of applications in biomedicine. In the case of applying diisocyanates for the crosslinking in water, it is necessary to show that the low molecular weight bifunctional crosslinker has fully reacted, while tailoring of the mechanical properties of the resulting hydrogels is possible despite the complex reaction mechanism. Here, the formation of gelatin-based hydrogel networks with the diisocyanates 2,4-toluene diisocyanate, 1,4-butane diisocyanate, and isophorone diisocyanate is presented. It is shown that extensive washing of materials is required to ensure full conversion of the diisocyanates. The use of different diisocyanates gives hydrogels covering a large range of Young’s moduli (12-450 kPa). The elongations at break (up to 83%) as well as the maximum tensile strengths (up to 410 kPa) of the hydrogels described here are much higher than for lysine diisocyanate ethyl ester crosslinked gelatin reported before. Rheological investigations suggest that the network formation in some cases is due to physical interactions and entanglements rather than covalent crosslink formation.

Gelatin can be covalently crosslinked in aqueous solution by application of diisocyanates like L-lysine diisocyanate ethyl ester in order to form hydrogels. Reaction of isocyanate groups with water is however a limiting factor in hydrogel network formation and can strongly influence the outcome of the crosslinking process. Here, diisocyanates with different water solubility and reactivity were applied for the formation of gelatin-based hydrogel networks and the mechanical properties of the hydrogels were investigated to gain a better understanding of starting material/ hydrogel property relations. L-Lysin diisocyanate ethyl ester (LDI), 2,4-toluene diisocyanate (TDI), 1,4-butane diisocyanate (BDI), and isophorone diisocyanate (IPDI) were selected, having different solubility in water ranging from 10-4 to 10-2 mol·L-1. BDI and LDI were estimated to have average reactive isocyanates groups, whereas TDI is highly reactive and IPDI has low reactivity. Formed hydrogels showed different morphologies and were partially very inhomogeneous. Gelation time (1 to 50 minutes), water uptake (300 to 900 wt.-%), and mechanical properties determined by tensile tests (E-moduli 35 to 370 kPa) and rheology (Shear moduli 4.5 to 19.5 kPa) showed that high water solubility as well as high reactivity leads to the formation of poorly crosslinked or inhomogeneous materials. Nevertheless, diisocyanates with lower solubility in water and low reactivity are able to form stable, homogeneous hydrogel networks with gelatin in water.

The formation of a functionally-confluent and shear-resistant endothelial cell (EC) monolayer on cardiovascular implants is a promising strategy to prevent thrombogenic processes after implantation. On the basis of existing studies with arterial endothelial cells adhering after two hours on gelatin-based hydrogels in marked higher numbers compared to tissue culture plates, we hypothesized that also venous endothelial cells (HUVEC) should be able to adhere and form an endothelial monolayer on these hydrogels after days. Furthermore, variation of the hydrogel composition, which slightly influences the materials elasticity and even more the degradation behaviour, should have no considerable effect on HUVEC. Therefore, the monolayer formation and shear resistance of HUVEC were explored on two gelatin-based hydrogels differing in their elasticity (Young's moduli between 35 and 55 kPa) in comparison to a positive control (HUVEC on glass cover slips) and a negative control (HUVEC on glass cover slips activated with interleukin-1β) after 9 days of culturing. HUVEC density after 9 days of culturing under static conditions was lower on the hydrogels compared to both controls (p

Preparation of three-dimensionally architectured porous biomaterials can be achieved in a one-step process by stabilizing gelatin with L-lysine diisocyanate ethyl ester (LDI) in water. The reaction of gelatin with LDI in presence of water leads to the formation of oligourea bridges between gelatin molecules and oligourea chains grafted on gelatin. The number and the length of the bridges, as well as of the grafted chains strongly depend on the concentration of the LDI used for the stabilization, and this has huge influence on the mechanical properties of the material on different hierarchical levels. Higher LDI concentrations yield materials with increased deformation resistance in tensile tests due to the higher number of covalent and physical netpoints in the material. However, mechanical properties determined on the micro-level by AFM indentation showed the opposite trend, i.e. a decrease of Young’s modulus with increasing LDI content. This was interpreted by a decreasing number of shorter oligourea bridges between gelatin chains with decreasing LDI content.

Purpose The aromatic compounds desaminotyrosine (DAT) and desaminotyrosyltyrosine (DATT) have been successfully used to functionalize gelatin in order to form physically crosslinked networks via p-p interactions and hydrogen bonds of the introduced phenol moieties. Here, it was explored whether this concept can be applied to a synthetic polymer not engaging in additional interactions such as triple helix formation in gelatin, enabling a network to form by physical interactions mainly related to the terminal functional groups. Oligo(ethylene glycol) (OEG) was chosen as hydrophilic synthetic polymer for the backbone structure. Methods Linear OEG (MP = 3 kDa) and four-arm OEG (Mn = 5 kDa) with amino functionalities as endgroups were functionalized with DAT and DATT using EDC·HCl and NHS as activating agents. The compounds were characterized by NMR, IR spectroscopy, and MALDI. Rheological behavior of aqueous solutions of the polymers was studied. The critical micelle concentration (CMC) was determined by a fluorescence spectroscopic analysis using the hydrophobic fluorescent dye pyrene. Results DATT-functionalized linear OEG, four-arm DAT-functionalized OEG and four-arm DATT-functionalized OEG were synthesized with degrees of functionalization of 60–95 mol%. All compounds were water soluble, and rheological measurements revealed a decrease in storage modulus G′ and loss modulus G″ compared to unfunctionalized OEG. Moreover, the CMC of linear OEG-DATT could be determined. Conclusions The syntheses of OEG functionalized with the aromatic compounds DAT and DATT was reported. The polymers showed the properties of a surfactant.

Biopolymer-based systems with adjustable macroscopic properties that can be varied in a wide range using only small changes in chemical composition are promising candidates for biomaterial-induced autoregeneration. Glycidylmethacrylated gelatin is photopolymerized with the addition of PEG mono- or dimethacrylate to form co-networks in pH = 7.4 PBS. The degree of swelling (Q) and water uptake (H) in PBS at 37 °C are tailorable for PEGDMA co-networks (Q ≈ 250-650 vol%), while the storage modulus of swollen networks at 3 °C can be adjusted by the PEG(D)MA content (G' = 0.7-145 kPa). Indirect cytotoxicity tests on ethylene oxide sterilized films show non-toxic responses for the homonetwork and all but one PEGDMA-containing co-networks materials.

Telechelic oligomers are attractive starting materials for the preparation of materials with tailorable properties. Here, peptide chains with defined molecular weight were obtained by controlled degradation of gelatin with hydroxylamine, which resulted in the cleavage of asparaginyl-glycine bonds and formation of new aspartyl hydroxamates and amino endgroups. The reaction of gelatin with hydroxylamine resulted in fragments with molecular weights of 15, 25, 37, and 50 kDa (determined by SDS-PAGE) independently of the reaction time and conditions. The fragment mixture showed typical single and triple helical organization in WAXS spectra, but rheological studies showed lower G′ and G″ values for gels from the fragment mixture than from gelatin, and lower gel-sol transitions temperatures. A more narrow distribution was found for the fragment mixture (M n = 25 kDa, PDI = 1.4) than for commercial gelatin. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biomaterial-induced autoregeneration requires materials with distinct tailored mechanical and thermal properties, water uptake and swelling properties as well as degradation behavior. Furthermore, before any biomaterial can be applied in vivo, in vitro studies should be performed that confirm the suitability for such applications. One facet in this process is the evaluation of endotoxin loads and immunogenic response to the material to avoid an unspecific activation of the immune system, which otherwise might cause fever and could lead to life–threatening pathologies. In this study, gelatins functionalized with desaminotyrosine (DAT) or desaminotyrosyl tyrosine (DATT) were investigated in terms of their endotoxin content and their potential to induce an inflammatory cytokine response in macrophages. Using the Limulus amebocyte lysate (LAL) test it could be shown that the endotoxin content was substantially reduced by using certified low endotoxin containing gelatin and performing the gelatin functionalization under cleanroom conditions. Furthermore, production of inflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor–alpha (TNFα) of an immune relevant macrophage cell line was significantly reduced for these materials. The survival of the macrophage cell line in the presence of DAT(T)-functionalized gelatins was not influenced by both materials. Therefore, DAT- and DATT-functionalized gelatins were shown to have passed the tests concerning immunological responses important for their applicability in vivo.

Molecular models of gelatin-based materials formed the basis for the knowledge-based design of a physically cross-linked polymer system. The computational models with 25 wt.-% water content were validated by comparison of the calculated structural properties with experimental data and were then used as predictive tools to study chain organization, cross-link formation, and estimation of mechanical properties. The introduced tyrosine-derived side groups, desaminotyrosine (DAT) and desaminotyrosyl tyrosine (DATT), led to the reduction of the residual helical conformation and to the formation of physical net-points by π-π interactions and hydrogen bonds. At 25 wt.-% water content, the simulated and experimentally determined mechanical properties were in the same order of magnitude. The degree of swelling in water decreased with increasing the number of inserted aromatic functions, while Young's modulus, elongation at break, and maximum tensile strength increased.

In the fields of surgery and regenerative medicine, it is crucial to understand the interactions of proteins with the biomaterials used as implants. Protein adsorption directly influences cell-material interactions in vivo and, as a result, regulates, for example, cell adhesion on the surface of the implant. Therefore, the development of suitable analytical techniques together with well-defined model systems allowing for the detection, characterization, and quantification of protein adsorbates is essential. In this study, a protocol for the deposition of highly stable, thin gelatin-based films on various substrates has been developed. The hydrogel films were characterized morphologically and chemically. Due to the obtained low thickness of the hydrogel layer, this setup allowed for a quantitative study on the interaction of human proteins (albumin and fibrinogen) with the hydrogel by Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). This technique enables the determination of adsorbant mass and changes in the shear modulus of the hydrogel layer upon adsorption of human proteins. Furthermore, Secondary Ion Mass Spectrometry and principal component analysis was applied to monitor the changed composition of the topmost adsorbate layer. This approach opens interesting perspectives for a sensitive screening of viscoelastic biomaterials that could be used for regenerative medicine.

Gelatins functionalized with desaminotyrosine (DAT) or desaminotyrosyl tyrosine (DATT) form physically crosslinked hydrogels, due to the interactions between the introduced aromatic moieties and gelatin triple helices, whose extent depends on the thermal treatment of the material. The G-modulus of these hydrogels can be tailored to the range of the natural extracellular matrix by adjusting the degree of crosslinking. While these gelatin-based materials have been shown to be not angiogenic, the aim of the study was to evaluate whether these biomaterials influence the regulation of blood vessels when positioned on the chorionallantoic membrane (CAM) of fertilized eggs. The results clearly indicate that the DAT-functionalized gelatin led to an increase of the diameter of the blood vessels in the CAM, which at the same time is probably associated with an increased blood flow in these CAM vessels. The vessel diameters of the four groups (DAT-functionalized gelatin, DATT-functionalized gelatin, plain gelatin, control group without gelatin, each n = 10) differed significantly (p0.0001). Vessels in the CAM exposed to the DAT-functionalized gelatin showed with 36.4 μm ± 3.4 μm the largest mean diameters compared to the mean diameters of the samples exposed to DATT gelatin (16.0 μm ± 0.8 μm; p0.05) and the plain gelatin (21.2 μm ± 1.0 μm; p0.05), which both did not differ significantly from the vessels of the control group. The biocompatibility of the materials in vitro motivates the exploration of their application as matrix in local drug-release systems with short half-life times (one hour up to several days).

Gelatins functionalized with desaminotyrosine or desaminotyrosyl tyrosine form physically crosslinked polymer networks due to the interactions between the introduced aromatic moeties. In the swollen state, their mechanical properties can be tailored in a range similar to the elasticity of soft tissues. The aim of this study was to evaluate their potential as biomaterials by determining whether these materials - in comparison to plain gelatin - induce bleedings, thrombotic processes, or angiogenesis. These investigations were performed using the hen's egg chorioallantoic membrane (HETCAM) assay. These results indicate that the gelatin-based hydrogels did not possess angiogenic effects and also did not induce bleedings, thrombotic processes or vessel destruction (avascular zones). The biocompatibility of the materials in vitro motivates the exploration of their application as matrix in local drug-release systems with short half-life times (1 hour up to several days).

Biomimetic polymer network systems with tailorable properties based on biopolymers represent a class of degradable hydrogels that provides sequences for protein adsorption and cell adhesion. Such materials show potential for in vitro MSC proliferation as well as in vivo applications and were obtained by crosslinking different concentrations of gelatin using varying amounts of ethyl lysine diisocyanate in the presence of a surfactant in pH 7.4 PBS solution. Material extracts, which were tested for cytotoxic effects using L929 mouse fibroblasts, were non-toxic. The hydrogels were seeded with human bone marrow-derived MSCs and supported viable MSCs for the incubation time of 9 d. Preadsorption of fibronectin on materials improved this biofunctionality.

Physically crosslinked hydrogels based on gelatin functionalized with desaminotyrosine (DAT) (giving Gel-DAT) or desaminotyrosyl tyrosine (DATT) (resulting in Gel-DATT) have shown high potential as biomaterials. Here, protein adsorption to the functionalized gelatins in comparison to gelatin was quantified to see if the functionalization and chain organization of gelatins has an influence on the amount of proteins being adsorbed. For this purpose, gelatin, Gel-DAT, and Gel-DATT were incubated with water or aq. solutions of bovine serum albumin (BSA), fibrinogen, or fibronectin, respectively, at physiological concentrations. Protein concentrations in the supernatant were determined with the bicinchoninic acid (BCA) assay before and after the contact. BSA adsorption to the materials was influenced as well by the hydrophobicity of the material as the degree of swelling, with the observation that higher protein concentrations led to lower protein adsorption. The highest amount of fibronectin was adsorbed to Gel-DAT, followed by gelatin and Gel-DATT, with only small differences for different initial protein concentrations. Fibrinogen adsorption increased with increasing concentration. In the future, adsorption studies based on specific antibody-based techniques might enable quantification of the proteins also in competition assays and direct quantification of adsorbed material.

Stimuli-sensitive materials can change properties upon exposure to an external stimulus. Thermoreversible gelation upon heating is one example for such a stimuli sensitivity. Here, it is of significance to tailor the transition temperature and to achieve large changes of G’ and the viscosity. Grafting of the thermosensitive poly(ethylene glycol-b-propylene glycol-b-ethylene glycol)s (PEPEs) to pectin was performed in order to investigate if tailoring of the sol-gel-transition temperature can be achieved by adjusting the grafting ratio. PEPEs were aminated and grafted to the polysaccharide via EDC coupling as shown by FTIR. The sol-gel transition of the pectin, PEPE, and the grafted system (PGP) was investigated by rheology. The gelation temperature (Tgel) of the system could be adjusted by varying the grafting density of PEPE onto pectin as well as by the concentration of the thermosensitive polymer in aqueous solution. A concentration of 15 – 20 wt% of the grafted system in water led to gelation temperatures in the range of 25 – 33 °C and the critical micelle concentration (CMC) and critical micelle temperature (CMT) of the grafted systems were determined by UV spectroscopy. The viscosity and the G’ increased by four orders of magnitudes at Tgel, which is comparable to PEPEs alone, but could be reached at lower PEPE concentrations. In the future, a thorough mechanistic investigation of the gelation process would be of interest.

The single and triple helical organization of protein chains strongly influences the mechanical properties of gelatin-based materials. A chemical method for obtaining different degrees of helical organization in gelatin is covalent functionalization, while a physical method for achieving the same goal is the variation of the drying conditions of gelatin solutions. Here we explored how the introduction of desaminotyrosine (DAT) and desaminotyrosyl tyrosine (DATT) linked to lysine residues of gelatin influenced the kinetics and thermodynamic equilibrium of the helicalization process of single and triple helices following different drying conditions. Drying at a temperature above. the helix-to-coil transition temperature of gelatin (T > T-c, called nu(short)) generally resulted in gelatins with relatively lower triple helical content (X-c,X-t = 1-2%) than lower temperature drying (T < T-c, called nu(long)) (X-c,X-t = 8-10%), where the DAT(T) functional groups generally disrupted helix formation. While different helical contents affected the thermal transition temperatures only slightly, the mechanical properties were strongly affected for swollen hydrogels (E = 4-13 kPa for samples treated by nu(long) and E = 120-700 kPa for samples treated by nu(short)). This study shows that side group functionalization and different drying conditions are viable options to control the helicalization and macroscopic properties of gelatin-based materials.

Biomaterials are used in regenerative medicine for induced autoregeneration and tissue engineering. This is often challenging, however, due to difficulties in tailoring and controlling the respective material properties. Since functionalization is expected to offer better control, in this study gelatin chains were modified with physically interacting groups based on tyrosine with the aim of causing the formation of physical crosslinks. This method permits application-specific properties like swelling and better tailoring of mechanical properties. The design of the crosslink strategy was supported by molecular dynamic (MD) simulations of amorphous bulk models for gelatin and functionalized gelatins at different water contents (0.8 and 25 wt.-%). The results permitted predictions to be formulated about the expected crosslink density and its influence on equilibrium swelling behavior and on elastic material properties. The models of pure gelatin were used to validate the strategy by comparison between simulated and experimental data such as density, backbone conformation angle distribution, and X-ray scattering spectra. A key result of the simulations was the prediction that increasing the number of aromatic functions attached to the gelatin chain leads to an increase in the number of physical netpoints observed in the simulated bulk packing models. By comparison with the Flory-Rehner model, this suggested reduced equilibrium swelling of the functionalized materials in water, a prediction that was subsequently confirmed by our experimental work. The reduction and control of the equilibrium degree of swelling in water is a key criterion for the applicability of functionalized gelatins when used, for example, as matrices for induced autoregeneration of tissues.

Combining gelatins functionalized with the tyrosine-derived groups desaminotyrosine or desaminotyrosyl tyrosine with hydroxyapatite (HAp) led to the formation of composite materials with much lower swelling ratios than those of the pure matrices. Shifts of the infra-red (IR) bands related to the free carboxyl groups could be observed in the presence of HAp, which suggested a direct interaction of matrix and filler that formed additional physical cross-links in the material. In tensile tests and rheological measurements the composites equilibrated in water had increased Young's moduli (from 200 kPa up to 2 MPa) and tensile strengths (from 57 kPa up to 1.1 MPa) compared with the matrix polymers without affecting the elongation at break. Furthermore, an increased thermal stability of the networks from 40 to 85 degrees C could be demonstrated. The differences in the behaviour of the functionalized gelatins compared with pure gelatin as a matrix suggested an additional stabilizing bond between the incorporated aromatic groups and the HAp as supported by the IR results. The composites can potentially be applied as bone fillers.

Gelatin is a non-immunogenic and degradable biopolymer, which is widely applied in the biomedical field e. g. for drug capsules or as absorbable hemostats. However, gelatin materials present limited and hardly reproducible mechanical properties especially in aqueous systems, particularly caused by the uncontrollable partial renaturation of collagen-like triple helices. Therefore, mechanically demanding applications for gelatin-based materials, such as vascular patches, i.e. hydrogel films that seal large incisions in vessel walls, and for induced autoregeneration, are basically excluded if this challenge is not addressed. Through the synthesis of a defined chemical network of gelatin with hexamethylene diisocyanate (HDI) in DMSO, the self-organization of gelatin chains could be hindered and amorphous gelatin films were successfully prepared having Young's moduli of 60-530 kPa. Transferring the crosslinking reaction with HDI and, alternatively, ethyl lysine diisocyanate (LDI), to water as reaction medium allowed the tailoring of swelling behaviour and mechanical properties by variation of crosslinker content while suppressing the formation of helices. The hydrogels had Young's moduli of 70-740 kPa, compressive moduli of 16-48 kPa, and degrees of swelling of 300-800 vol%. Test reactions investigated by ESI mass spectrometry allowed the identification and quantification of reaction products of the crosslinking reaction. The HDI crosslinked networks were stabilized by direct covalent crosslinks (ca. 10 mol%), supported by grafting (50 mol%) and blending of hydrophobic oligomeric chains. For the LDI- based networks, less crosslinked (3 mol%) and grafted species (5 mol%) and much higher amounts of oligomers were observed. The adjustable hydrogel system enables the application of gelatin-based materials in physiological environments.

Gelatin was functionalized with glycidyl methacrylate and photocrosslinked in the presence of poly(ethylene glycol) dimethacrylate (PEGDMA) or poly(ethylene glycol) monomethacrylate (PEGMA) to create a biopolymer-based system with tailorable properties. These co-networks were hydrolyzed using 6 M HCl and the degradation products were analyzed and identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. This technique successfully identified gelatin-derived peptides such as FLPEPPE, SFLPEPPE, and SFLPEPPEE as well as an accompanying PEG-g-poly(methacrylic acid) component. No oligo- or polymethacrylates were monitored at any molecular weight range above m/z = 500, which indicated that they possessed lower molecular weights. An in vitro hydrolytic degradation experiment performed in pH 7.4 PBS buffer solution at 37 °C showed that these networks, which were prepared without the addition of a potentially toxic photoinitiator, exhibited mass loss of up to 50 wt% at 6 weeks of incubation time. These results provide valuable insight into how these functional gelatin-based co-network biomaterials will perform in a biological setting.