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The major challenge in liver tissue engineering is the replication of the microenvironment and microarchitecture of the liver tissue at the nanoscale. Decellularized liver matrix (DLM) provides an ideal material for scaffold preparation, as it retains the relevant structural and biochemical composition. However, the loss of bioactive factors during decellularization needs to be taken into account when using DLM and should be supplemented accordingly for an expected outcome. This study reports on the modification of DLM by the addition of galactose residues using a two-step thiol-ene-mediated photoclick chemistry for the coupling of galactose moieties to the DLM. Modification with galactose enhanced the function of hepatocytes and provides many advantages over currently used DLM and DLM-based materials. The galactose modified DLM enhanced the initial HepG2 cell adhesion to the substrate with changes in dynamics over time such as spheroid formation and further migration on the matrix. Our observation is that the galactose ligand decoration can also enhance the liver-specific metabolism of HepG2 compared to unmodified DLM. Galactosylated DLM also showed a better establishment of cellular polarity which also contributes to the function of HepG2 cells. Together our results demonstrate the advantages of adding galactose residues to currently available biomaterials, which makes this approach an attractive method for ECM-based liver tissue engineering.

Hydrogels are useful platforms as three-dimensional (3D) scaffolds for cell culture, drug-release systems, and regenerative medicine applications. Here, we propose a novel chemical cross-linking approach by the use of 3,4-diethoxy-3-cyclobutene-1,2-dione or diethyl squarate for the preparation of 5 and 10% w/v gelatin-based hydrogels. Hydrogels showed good swelling properties, and the 5% gelatin-based hydrogel proved suitable as a 3D cell culture scaffold for the chondrocyte cell line C28/I2. In addition, diffusion properties of different sized molecules inside the hydrogel were determined.

This review aims at giving selected chemical and mechanical insights on design criteria that should be taken into account in hydrogel production for biomedical applications. Particular emphasis will be given to the chemical aspects involved in hydrogel design: macromer chemical composition, cross-linking strategies and chemistry towards "conventional" and smart/stimuli responsive hydrogels. Mechanical properties of hydrogels in view of regenerative medicine applications will also be considered.

Binary nano-biocomposite 3D scaffolds of cellulose nanocrystals (CNCs) - gelatin were fabricated without the use of chemical cross-linking additives. Controlled oxidative treatment allowed the introduction of carboxyl or carbonyl functionalities on the surface of CNCs that were responsible for the cross-linking of gelatin polymers. The obtained composites were characterized for their physico-chemical properties, and their biocompatibility towards different cell cultures was eval-uated through MTT and LDH assays, cellular adhesion and proliferation experiments. Gelatin composites reinforced with carbonyl-modified CNCs showed the most performing swell-ing/degradation profile and the most promising adhesion and proliferation properties towards cell lines, suggesting their potential application in the field of tissue engineering

The present study reports on the synthesis of a new alkoxysilane-bearing light-responsive cinnamyl group and its application as a surface functionalization agent for the development of SiO2 nanoparticles (NPs) with photoreversible tails. In detail, cinnamic acid (CINN) was activated with N-hydroxysuccinimide (NHS) to obtain the corresponding NHS-ester (CINN-NHS). Subsequently, the amine group of 3-aminopropyltriethoxysilane (APTES) was acylated with CINN-NHS leading to the generation of a novel organosilane, CINN-APTES, which was then exploited for decorating SiO2 NPs. The covalent bond to the silica surface was confirmed by solid state NMR, whereas thermogravimetric analysis unveiled a functionalization degree much higher compared to that achieved by a conventional double-step post-grafting procedure. In light of these intriguing results, the strategy was successfully extended to naturally occurring sepiolite fibers, widely employed as fillers in technological applications. Finally, a preliminary proof of concept of the photoreversibility of the obtained SiO2@CINN-APTES system has been carried out through UV diffuse reflectance. The overall outcomes prove the consistency and the versatility of the methodological protocol adopted, which appears promising for the design of hybrid NPs to be employed as building blocks for photoresponsive materials with the ability to change their molecular structure and subsequent properties when exposed to different light stimuli.

Gelatin is a costless polypeptide material of natural origin, able to form hydrogels that are potentially useful in biomaterial scaffold design for drug delivery, cell cultures, and tissue engineering. However, gelatin hydrogels are unstable at physiological conditions, losing their features only after a few minutes at 37 °C. Accordingly, treatments to address this issue are of great interest. In the present work, we propose for the first time the use of bi- and trifunctional tetrazoles, most of them unknown to date, for photoinduced gelatin cross-linking towards the production of physiologically stable hydrogels. Indeed, after UV-B irradiation, aryl tetrazoles generate a nitrilimine intermediate that is reactive towards different functionalities, some of them constitutively present in the amino acid side chains of gelatin. The efficacy of the treatment strictly depends on the structure of the cross-linking agent used, and substantial improved stability was observed by switching from bifunctional to trifunctional cross-linkers.

In order to improve collagen bioactivity for regenerative medicine approaches, thymosin-β4 (Tβ4P) and Human Vitronectin (HVP) derived peptides are grafted to collagen by thiol-ene Michael addition. Tβ4P and HVP are known to exert a pro-angiogenic and a pro-adhesive activity respectively and HVP is involved in osteogenesis promotion. The ability of these peptides to increase collagen cell adhesion and angiogenesis properties is assessed on human cell lines. In particular, HVP-grafted collagen increased human osteoblast adhesion and cell proliferation: after 24 h, both adhesion and proliferation roughly showed a 4-fold increase, if compared to pristine collagen. Tβ4P-grafted collagen promotes Vascular Endothelial Growth Factor (VEGF) gene expression in human vascular cell lines by more than 7 times. These results suggest that HVP-grafted collagen may be an interesting biomaterial for bone tissue regeneration, while Tβ4P-grafted collagen is useful for angiogenesis promotion.

In lung cancer, CD133+ cells represent the subset of cancer stem cells (CSC) able to sustain tumor growth and metastatic dissemination. CSC function is tightly regulated by specialized niches composed of both stromal cells and extracellular matrix (ECM) proteins, mainly represented by collagen. The relevance of collagen glycosylation, a fundamental post‐translational modification controlling several biological processes, in regulating tumor cell phenotype remains, however, largely unexplored. To investigate the bioactive effects of differential ECM glycosylation on lung cancer cells, we prepared collagen films functionalized with glucose (Glc‐collagen) and galactose (Gal‐collagen) exploiting a neoglycosylation approach based on a reductive amination of maltose and lactose with the amino residues of collagen lysines. We demonstrate that culturing of tumor cells on collagen determines a glycosylation‐dependent positive selection of CSC and triggers their expansion/generation. The functional relevance of CD133+ CSC increase was validated in vivo, proving an augmented tumorigenic and metastatic potential. High expression of integrin β1 in its active form is associated with an increased proficiency of tumor cells to sense signaling from glycosylated matrices (glyco‐collagen) and to acquire stemness features. Accordingly, inhibition of integrin β1 in tumor cells prevents CSC enrichment, suggesting that binding of integrin β1 to Glc‐collagen subtends CSC expansion/generation. We provide evidence suggesting that collagen glycosylation could play an essential role in modulating the creation of a niche favorable for the generation and selection/survival of lung CSC. Interfering with this crosstalk may represent an innovative therapeutic strategy for lung cancer treatment., Glycans are well known for their involvement in recognition phenomena between cells; however, the role of small glycan epitopes in cell–extracellular matrix (ECM) interactions needs to be further elucidated. Here we exploit bioactive ECM mimetics, functionalizing type I collagen films with different glycans, to investigate the relevance of specific ECM glycosignatures in tumor‐ECM interactions. We show that in vitro culturing of lung cancer cells on glycosylated collagen films results in differential modulation of cancer stem cells (CSC), associated in vivo with an enhanced tumor initiation ability and increased metastatic potential. Interactions of CSC with glycosylated collagen are regulated through the active form of integrin β1. Interfering with integrin β1 signaling results in abrogation of CSC enrichment induced by glycosylated collagen.

The determination of the adipogenic potential and behavior of adipose-derived mesenchymal stem/stromal cells (ASCs) is particularly relevant for their potential clinical application in regenerative medicine, especially when the regeneration processes is supported by biomaterials or scaffolds. Scaffolds should be able to induce tissue repair, limiting the undesired adipogenic differentiation. Depending on the scaffold, the determination of cell behavior may be hindered, due to material interference with staining limiting either cells identification or dye quantification. Collagen is a promising biomaterial in regenerative medicine, however histological analysis of cells cultured on collagen-based scaffolds is challenging. Herein we propose a new histological method, based on the combination of iron hematoxylin and Oil Red O (ORO) staining, for the determination of the adipogenic differentiation of ASCs cultivated on a collagen-based 2D scaffold. ASCs were seeded on collagen films or plastic, differentiated into adipocytes for 14 days, then stained either with ORO or with a combination of iron hematoxylin and ORO. As the collagen films highly absorb the ORO dye, conventional staining and quantification by dye extraction did not allow to discriminate between differentiated and undifferentiated cells on collagen films. On the contrary, iron hematoxylin-ORO combination allowed a quantitative and more reliable determination of adipocytes based on single cell count. The proposed method is particularly recommended for the determination of adipogenic differentiation potential of ASCs and other cell types when grown on highly absorptive materials that need to be validated for their potential use in bioengineering and regenerative medicine.

The ability of gelatin-based hydrogels of incorporating and releasing under controlled conditions 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a melanin-related metabolite endowed with marked antioxidant properties was investigated. The methyl ester of DHICA, MeDHICA, was also tested in view of its higher stability, and different solubility profile. Three types of gelatin-based hydrogels were prepared: pristine porcine skin type A gelatin (HGel-A), a pristine gelatin cross-linked by amide coupling of lysines and glutamic/aspartic acids (HGel-B), and a gelatin/chitosan blend (HGel-C). HGel-B and HGel-C differed in the swelling behavior, showed satisfactorily high mechanical strength at physiological temperatures and well-defined morphology. The extent of incorporation into all the gelatins tested using a 10% w/w indole to gelatin ratio was very satisfactory ranging from 60 to 90% for either indoles. The kinetics of indole release under conditions of physiological relevance was evaluated up to 72 h. The highest values were obtained with HGel-B and HGel-C for MeDHICA (90% after 6 h), and an appreciable release was observed for DHICA reaching 30% and 40% at 6 h for HGel-B and HGel-C, respectively. At 72 h, DHICA and MeDHICA were released at around 30% from HGel-A at pH 7.4, with an increase up to 40% at pH 5.5 in the case of DHICA. DHICA incorporated into HGel-B proved fairly stable over 6 h whereas the free compound at the same concentration was almost completely oxidized. The antioxidant power of the indole loaded gelatins was monitored by chemical assays and proved unaltered even after prolonged storage in air, suggesting that the materials could be prepared in advance with respect to their use without alteration of their efficacy.

Glycans influence a variety of aspects of tissue development and pathophysiology, harboring a wealth of biochemical information exploitable for the discovery of new biomarkers. The development of versatile and precise glycoanalytical platforms is crucial to bring to the scientific community reliable tools to unveil the glycan- encoded biochemical information. In this work, a novel platform for brain glycan analysis, named Lysate in-Solution Deglycosylation (LSD), is presented, validated and compared to similar methodologies reported in the literature. Briefly, within the LSD workflow, brain tissue is lysed, proteins are purified via methanol/chloroform extraction, and directly deglycosylated with PNGase F. Released N- glycans are purified using a second round of methanol/chloroform extraction, labelled with fluorescent tags, and cleaned-up prior to LC- FLR/MS analysis. LSD is a quick, sensitive, robust, precise, specific and versatile glycomic workflow, compatible with multiple glyco(proteo)mics analyses of the same sample and eventually applicable to other solid tissues. LSD was devised as a plug-and-play platform for quick glycosylation analysis, and was designed to be compatible with a variety of analytical set-ups (i.e., diverse detection systems), thus enabling the extraction of an additional layer of information from different studies (i.e., proteomics) without the need to collect fresh biological materials. In comparison to analogous N-glycomic workflows, LSD performs reliably on the smallest amount of starting brain tissue and, to the best of the authors' knowledge, is currently the most thoroughly validated neuro-N- glycomic method in the field-related literature, thus representing a significant step forward on the road towards glycan-based biomedical applications.

Palmitoylethanolamide (PEA) belongs to the class of N-acylethanolamine and is an endogenous lipid potentially useful in a wide range of therapeutic areas, products containing PEA are licensed for use in humans as a nutraceutical, a food supplement, or food for medical purposes for its analgesic and anti-inflammatory properties demonstrating efficacy and tolerability. However, the exogenously administered PEA is rapidly inactivated, in this process, fatty acid amide hydrolase (FAAH) plays a key role both in hepatic metabolism and in intracellular degradation. So, the aim of the present study was the design and synthesis of PEA analogues that are more resistant to FAAH-mediated hydrolysis. A small library of PEA analogues was designed and tested by molecular docking and density functional theory calculations to find the more stable analogue. The computational investigation identified RePEA as the best candidate in terms of both synthetic accessibility and metabolic stability to FAAH-mediated hydrolysis. The selected compound was synthesized and assayed ex vivo to monitor FAAH-mediated hydrolysis and to confirm its anti-inflammatory properties. 1H-NMR spectroscopy performed on membrane samples containing FAAH in integral membrane protein demonstrated that RePEA is not processed by FAAH, in contrast with PEA. Moreover, RePEA retains PEA&rsquo, s ability to inhibit LPS-induced cytokine release in both murine N9 microglial cells and human PMA-THP-1 cells.

The Nobel 2018 goes to the evolution of chemistry with biotechnologies. The Nobel Prize 2018 in Chemistry went to scientists who developed biotechnological tools for accelerating and controlling in vitro the evolution of proteins. These methodologies, conceived by Arnold, Winter and Smith, have delivered biocatalysts of practical utility for the chemical industry but also a new generation of therapeutic agents for cancer and immunological diseases.

In the last few decades, tremendous progresses have been achieved in the field of regenerative medicine, as a consequence of the better understanding of cell biology, the advances in material science, chemistry, and engineering, and the integration of all of these disciplines within a highly synergistic and multidisciplinary context. Within this extremely multidisciplinary and interdisciplinary field, carbohydrates can play key roles, from being materials that can be shaped into scaffolds able to sustain cell adhesion and proliferation, to signaling biocues capable to drive cell differentiation or homing: carbohydrates chemistry and biology may open new avenues in regenerative medicine. In this chapter, carbohydrates will be presented as biomaterials offering a wide range of useful mechanical and biological properties, as bioactivating cues of materials of different chemical nature and finally as targets of cell surface engineering strategies able to modulate cell fate, in view of advanced cell therapies applications.

Tissue engineering relies on the possibility to engineer cell microenvironments by means of bioactive materials, biochemical and physical stimuli in order to guide cell behaviour and to regenerate damaged tissue. Despite the relevance of glycan epitopes as signaling molecules, and the recent advances in glycomics, their use as biomolecular cues at the interface between materials and cells for the controlled stimulation of adhesion and differentiation processes for regenerative medicine applications is still limited. In this concept article we will briefly outline the basis and the impact on health and economics of regenerative medicine, together with the recent applications of the glycocode in tissue regeneration approaches.

Carbohydrates are one of the most powerful and versatile classes of biomolecules that nature uses to regulate organisms’ biochemistry, modulating plenty of signaling events within cells, triggering a plethora of physiological and pathological cellular behaviors. In this framework, glycan carrier systems or carbohydrate-decorated materials constitute interesting and relevant tools for medicinal chemistry applications. In the last few decades, efforts have been focused, among others, on the development of multivalent glycoconjugates, biosensors, glycoarrays, carbohydrate-decorated biomaterials for regenerative medicine, and glyconanoparticles. This review aims to provide the reader with a general overview of the different carbohydrate carrier systems that have been developed as tools in different medicinal chemistry approaches relying on carbohydrate-protein interactions. Given the extent of this topic, the present review will focus on selected examples that highlight the advancements and potentialities offered by this specific area of research, rather than being an exhaustive literature survey of any specific glyco-functionalized system.

The emerging trends in regenerative medicine rely among others on biomaterial-based therapies, with the use of biomaterials as a central delivery system for biochemical and physical cues to manipulate transplanted or ingrowth cells and to orchestrate tissue regeneration. Cell adhesion properties of a biomaterial strongly depend on its surface characteristics. Among others poly(e-caprolactone) (PCL) is a biocompatible and biodegradable material with low cytotoxicity that is widely adopted as synthetic polymer in several applications. However, it is hydrophobic, which limits its use in tissue engineering. In order to improve its hydrophilicity and cellular compatibility, PCL surface was grafted with maltose through a two-step procedure in which controlled aminolysis of PCL ester bonds by hexanediamine was followed by reductive amination with the carbohydrate reducing end. The modified PCL surface was then characterized in detail by x-ray Photoelectron Spectroscopy (XPS) and Near Edge x-ray Absorption Fine Structure (NEXAFS) spectroscopies. In addition, the biocompatibility of the proposed biomaterial was investigated in preliminary biological assays.

cited By 1; International audience; Conventional composites often do not represent true synergy of their constituent materials. This is particularly evident in biomaterial applications where devices must interact with cells, resist cyclic loads and biodegrade safely. Here we propose a new hybrid system, with co-networks of organic and inorganic components, resulting in unprecedented mechanical properties, including "bouncy" elasticity and intrinsic ability to self-heal autonomously. They are also developed as new 'inks' that can be directly 3D printed. A hybrid is different from a nanocomposite because the components are indistinguishable from each other at the nanoscale and above. The properties are generated by a novel methodology that combines in situ cationic ring-opening polymerisation with sol-gel, creating silica/poly(tetrahydrofuran)/poly(ϵ-caprolactone) hybrids with molecular scale interactions and covalent links. Cartilage is notoriously difficult to repair and synthetic biomaterials have yet to mimic it closely. We show that 3D printed hybrid scaffolds with pore channels of ∼200 μm mimic the compressive behaviour of cartilage and provoke chondrocytes to produce markers integral to articular cartilage-like matrix. The synthesis method can be applied to different organic sources, leading to a new class of hybrid materials.

A new hydrogel, totally based on gelatin has been synthesized by cross-linking thiolated gelatin with pentenoyl gelatin, via thiol-ene click chemistry. Preliminary cytocompatibility assays with hBMSC showed good cell compatibility.

New glucuronosyldiacylglycerol (GlcADG) analogues based on a 2-O-β-d-glucopyranosyl-sn-glycerol scaffold and carrying one or two acyl chains of different lengths have been synthesized as phosphatidylinositol 3-phosphate (PI3P) mimics targeting the protein kinase Akt. The Akt inhibitory effect of the prepared compounds was assayed using an in vitro kinase assay. The antiproliferative activity of the compounds was tested in the human ovarian carcinoma IGROV-1 cell line in which we found that two of them could inhibit proliferation, in keeping with the target inhibitory effect.

The key signalling roles played by glycans (simple or complex) in the development of smart biomaterials for neuroregeneration is reported. The glycans involved and their diversity (glycomic), the nature and properties of biomaterials, the bioconjugation methods, and the effects in neuroregeneration are discussed.

The synthesis of novel hyperbranched monodisperse linear dendrimers, based on 2,2-bis-(hydroxymethyl)-propionic acid (bis-MPA), has been achieved by convergent metathesis-mediated coupling between the alkene-terminated focal point of bis-MPA dendrons. On their surface, dendrimers present 4, 8 and 16 functional groups. Glycodendrimers exposing multiple saccharide moieties have also been obtained. To the best of our knowledge, this is the first example of the use of metathesis for focal point coupling.

Hybrid materials, with co-networks of organic and inorganic components, are increasing in popularity due to their tailorable degradation rates and mechanical properties. To increase mechanical stability, particularly in water, covalent bonding must occur between the components. This can be introduced using crosslinking agents such as 3-glycidoxypropyl trimethoxysilane (GPTMS). Attachment of GPTMS to polymers in aqueous conditions is hypothesized to occur by opening of the epoxide ring by nucleophiles on the polymer chain. Despite side reactions that occur between the epoxide ring of GPTMS and water, a range of NMR techniques showed that the carboxylic acid group of poly(γ-glutamic acid) reacted with GPTMS. This result was used to identify the amino acids in gelatin that reacted most rapidly with the GPTMS epoxide ring, confirming that covalent bonding occurred in gelatin–silica hybrid materials.

The ability to replicate in vitro the native extracellular matrix (ECM) features and to control the three-dimensional (3D) cell organization plays a fundamental role in obtaining functional engineered bioconstructs. In tissue engineering (TE) applications, hydrogels have been successfully implied as biomatrices for 3D cell embedding, exhibiting high similarities to the natural ECM and holding easily tunable mechanical properties. In the present study, we characterized a promising photocrosslinking process to generate cell-laden methacrylate gelatin (GelMA) hydrogels in the presence of VA-086 photoinitiator using a ultraviolet LED source. We investigated the influence of prepolymer concentration and light irradiance on mechanical and biomimetic properties of resulting hydrogels. In details, the increasing of gelatin concentration resulted in enhanced rheological properties and shorter polymerization time. We then defined and validated a reliable photopolymerization protocol for cell embedding (1.5% VA-086, LED 2 mW/cm2) within GelMA hydrogels, which demonstrated to support bone marrow stromal cells viability when cultured up to 7 days. Moreover, we showed how different mechanical properties, derived from different crosslinking parameters, strongly influence cell behavior. In conclusion, this protocol can be considered a versatile tool to obtain biocompatible cell-laden hydrogels with properties easily adaptable for different TE applications.

New sulfoquinovose analogues of phosphatidylinositol 3-phosphate have been synthesised based on a sulfoquinovose scaffold as potential protein kinase B (PKB) inhibitors. The synthetic strategy involved the introduction into glucose of a thioacetate group at the 6-position and of an azide group at the anomeric position as precursors of the sulfonate and phosphoramidate moieties present in the final compounds. The synthesised compounds were tested in vitro on isolated PKB by means of ELISA assays and for their anti-proliferative activity against the human ovarian carcinoma cell line IGROV-1. Sulfoquinovose derivatives 2b and 2c showed inhibitory activity in the low micromolar range.

The synthesis of new dendrons of the generations 0, 1 and 2 with a double bond at the focal point and a carbonyl group at the termini has been carried out. The carbonyl group has been exploited for the multivalent conjugation to a sample saccharide by reductive amination and alkoxyamine conjugation. © 2014 Bini et al; licensee Beilstein-Institut.

Collagen matrices have been neoglycosylated with lactose by reductive amination at lysine side chains. AFM analysis highlights that the chemical neoglycosylation does not affect molecular assembly into fibrils. Moreover, ELLA biochemical assays show that the glycan moiety is efficiently exposed on the matrix surface for receptor recognition.

A set of new metabolically stable arabinose 5-phosphate analogues possessing phosphate mimetic groups at position 5 was synthesised. Their ability to interact with arabinose 5-phosphate isomerase from Pseudomonas aeruginosa was evaluated by STD-NMR studies. The synthesised compounds were also characterised for their activity in vivo on P. aeruginosa and Escherichia coli strains. Unfortunately, none of the synthesised compounds was able neither to bind API nor to inhibit bacterial growth.

Despite the relevance of carbohydrates as cues in eliciting specific biological responses, glycans have been rarely exploited in the study of neuronal physiology. We report thereby the study of the effect of neoglucosylated collagen matrices on neuroblastoma F11 cell line behavior. Morphological and functional analysis clearly showed that neoglucosylated collagen matrices were able to drive cells to differentiate. These data show for the first time that F11 cells can be driven from proliferation to differentiation without the use of chemical differentiating agents. Our work may offer to cell biologists new opportunities to study neuronal cell differentiation mechanisms in a cell environment closer to physiological conditions.

Biomaterial surface properties, via introduction of specific signal biomolecules can influence cell adhesion and differentiation processes. Inspired by the monosaccharide motifs found in collagen glycosylation patterns, galactose being one of the most commonly found saccharidic residues, collagen matrices were modified in order to expose galactose residues to cells. The interaction of chemically neoglycosylated matrices was evaluated with osteosarcoma-derived cell line MG63.

Hybrid materials with interpenetrating networks of silica and degradable polymers have the potential to outperform current biomaterials as their mechanical properties and degradation rates can be tightly controlled. GPTMS is one of the most widely used precursors for sol–gel hybrid fabrication. It can be used as the silica precursor or as a coupling agent to create hybrids with covalent bonds between the silica and organic components. Understanding its reactivity with nucleophilic groups on organic molecules in aqueous conditions is of key importance to hybrid synthesis. NMR spectrometry assisted by mass spectrometry was successfully used for the investigation of the reaction system of (3-glycidoxypropyl)trimethoxysilane (GPTMS) in the presence of different simple nucleophiles. Inorganic condensation and silica network formation is accelerated in the presence of propylamine; propanoic acid gives nucleophilic attack on the epoxy ring; propanol and propanthiol do not seem to participate in the reaction.

Gelatin is a biopolymer with interesting properties that can be useful for biomaterial design for different applications such as drug delivery systems, or 3D scaffolds for tissue engineering. However, gelatin suffers from poor mechanical stability at physiological temperature, hence methods for improving its properties are highly desirable. In the present work, a new chemical cross-linking strategy based on triazolinedione ene-type chemistry towards stable hydrogel is proposed. Two different homobifunctional 1,2,4-triazoline-3,5(4H)-diones, namely 4,4&prime, hexane-1,6-diylbis(3H-1,2,4-triazoline-3,5(4H)-dione) 1 and 4,4&prime, [methylenebis(4,1-phenylene)]bis(3H-1,2,4-triazoline-3,5(4H)-dione) 2 were used as cross-linkers in different ratio to tyrosine residues in gelatin. The reaction was proved effective in all experimented conditions and hydrogels featured with different thermal stability were obtained. In general, the higher the cross-linker/tyrosine ratio, the more thermostable the hydrogel. The swelling properties are strictly dependent upon the chemical nature of the cross-linker.