TEMPO/TCC as a Chemo Selective Alternative for the Oxidation of Hyaluronic Acid.

Hyaluronic acid (HA)-based hydrogels are very commonly applied as cell carriers for different approaches in regenerative medicine. HA itself is a well-studied biomolecule that originates from the physiological extracellular matrix (ECM) of mammalians and, due to its acidic polysaccharide structure, offers many different possibilities for suitable chemical modifications which are necessary to control, for example, network formation. Most of these chemical modifications are performed using the free acid function of the polymer and, additionally, lead to an undesirable breakdown of the biopolymer's backbone. An alternative modification of the vicinal diol of the glucuronic acid is oxidation with sodium periodate to generate dialdehydes via a ring opening mechanism that can subsequently be further modified or crosslinked via Schiff base chemistry. Since this oxidation causes a structural destruction of the polysaccharide backbone, it was our intention to study a novel synthesis protocol frequently applied to selectively oxidize the C6 hydroxyl group of saccharides. On the basis of this TEMPO/TCC oxidation, we studied an alternative hydrogel platform based on oxidized HA crosslinked using adipic acid dihydrazide as the crosslinker.

Appreciating the First Line of the Human Innate Immune Defense: A Strategy to Model and Alleviate the Neutrophil Elastase-Mediated Attack toward Bioactivated Biomaterials.

Biointerface engineering is a wide-spread strategy to improve the healing process and subsequent tissue integration of biomaterials. Especially the integration of specific peptides is one promising strategy to promote the regenerative capacity of implants and 3D scaffolds. In vivo, these tailored interfaces are, however, first confronted with the innate immune response. Neutrophils are cells with pronounced proteolytic potential and the first recruited immune cells at the implant site; nonetheless, they have so far been underappreciated in the design of biomaterial interfaces. Herein, an in vitro approach is introduced to model and analyze the neutrophil interaction with bioactivated materials at the example of nano-bioinspired electrospun surfaces that reveals the vulnerability of a given biointerface design to the contact with neutrophils. A sacrificial, transient hydrogel coating that demonstrates optimal protection for peptide-modified surfaces and thus alleviates the immediate cleavage by neutrophil elastase is further introduced.

Influence of Microgel Fabrication Technique on Granular Hydrogel Properties.

Bulk hydrogels traditionally used for tissue engineering and drug delivery have numerous limitations, such as restricted injectability and a nanoscale porosity that reduces cell invasion and mass transport. An evolving approach to address these limitations is the fabrication of hydrogel microparticles (i.e., "microgels") that can be assembled into granular hydrogels. There are numerous methods to fabricate microgels; however, the influence of the fabrication technique on granular hydrogel properties is unexplored. Herein, we investigated the influence of three microgel fabrication techniques (microfluidic devices (MD), batch emulsions (BE), and mechanical fragmentation by extrusion (EF)) on the resulting granular hydrogel properties (e.g., mechanics, porosity, and injectability). Hyaluronic acid (HA) modified with various reactive groups (i.e., norbornenes (NorHA), pentenoates (HA-PA), and methacrylates (MeHA)) were used to form microgels with an average diameter of ∼100 µm. The MD method resulted in homogeneous spherical microgels, the BE method resulted in heterogeneous spherical microgels, and the EF method resulted in heterogeneous polygonal microgels. Across the various reactive groups, microgels fabricated with the MD and BE methods had lower functional group consumption when compared to microgels fabricated with the EF method. When microgels were jammed into granular hydrogels, the storage modulus (G') of EF granular hydrogels (∼1000-3000 Pa) was consistently an order of magnitude higher than G' for MD and BE granular hydrogels (∼50-200 Pa). Void space was comparable across all groups, although EF granular hydrogels exhibited an increased number of pores and decreased average pore size when compared to MD and BE granular hydrogels. Furthermore, granular hydrogel properties were tuned by varying the amount of cross-linker used during microgel fabrication. Lastly, granular hydrogels were injectable across formulations due to their general shear-thinning and self-healing properties. Taken together, this work thoroughly characterizes the influence of the microgel fabrication technique on granular hydrogel properties to inform the design of future systems for biomedical applications.

Tuning the Product Spectrum of a Glycoside Hydrolase Enzyme by a Combination of Site-Directed Mutagenesis and Tyrosine-Specific Chemical Modification.

Selective chemical modification of proteins plays a pivotal role for the rational design of enzymes with novel and specific functionalities. In this study, a strategic combination of genetic and chemical engineering paves the way for systematic construction of biocatalysts by tuning the product spectrum of a levansucrase from Bacillus megaterium (Bm-LS), which typically produces small levan-like oligosaccharides. The implementation of site-directed mutagenesis followed by a tyrosine-specific modification enabled control of the product synthesis: depending on the position, the modification provoked either enrichment of short oligosaccharides (up to 800 % in some cases) or triggered the formation of high molecular weight polymer. The chemical modification can recover polymerization ability in variants with defective oligosaccharide binding motifs. Molecular dynamic (MD) simulations provided insights into the effect of modifying non-native tyrosine residues on product specificity.

Product-oriented chemical surface modification of a levansucrase (SacB) via an ene-type reaction.

Carbohydrate processing enzymes are sophisticated tools of living systems that have evolved to execute specific reactions on sugars. Here we present for the first time the site-selective chemical modification of exposed tyrosine residues in SacB, a levansucrase from Bacillus megaterium (Bm-LS) for enzyme engineering purposes via an ene-type reaction. Bm-LS is unable to sustain the synthesis of high molecular weight (HMW) levan (a fructose polymer) due to protein-oligosaccharide dissociation events occurring at an early stage during polymer elongation. We switched the catalyst from levan-like oligosaccharide synthesis to the efficient production of a HMW fructan polymer through the covalent addition of a flexible chemical side-chain that fluctuates over the central binding cavity of the enzyme preventing premature oligosaccharide disengagement.

Evaluation of Hydrogels Based on Oxidized Hyaluronic Acid for Bioprinting.

In this study, we evaluate hydrogels based on oxidized hyaluronic acid, cross-linked with adipic acid dihydrazide, for their suitability as bioinks for 3D bioprinting. Aldehyde containing hyaluronic acid (AHA) is synthesized and cross-linked via Schiff Base chemistry with bifunctional adipic acid dihydrazide (ADH) to form a mechanically stable hydrogel with good printability. Mechanical and rheological properties of the printed and casted hydrogels are tunable depending on the concentrations of AHA and ADH cross-linkers.

Comparison of two laboratory extraction techniques for the detection of Epstein-Barr virus in the saliva of nasopharyngeal carcinoma patients.

AIM: The aim of the present study was to compare the effectiveness of DNA extraction using an extraction kit against the standard boiling technique for the detection of Epstein-Barr virus (EBV) DNA in nasopharyngeal carcinoma (NPC) patients. METHODS: Stimulated whole saliva samples from newly-diagnosed NPC patients were collected. EBV DNA was extracted by both techniques (n = 23) followed by quantitative real-time polymerase chain reaction (PCR) using the primer/probe set for BALF5. RESULTS: The results of the quantitative real-time PCR were reproducible in both groups. The two techniques were moderately correlated (r = 0.67, P < 0.05), and the degree of agreement was good. However, the mean EBV DNA level in the boiling group (3.02 ± 8.67 × 10(6) copies/µL) was significantly higher than the extraction kit group (1.15 ± 2.66 × 10(6) copies/µL) (P < 0.05). The EBV DNA level was higher in patients at an advanced overall stage (P = 0.05). CONCLUSION: The results of the present study showed that the performance of the extraction kit was not superior to the simple boiling technique for the detection of salivary EBV DNA in NPC patients using real-time PCR. The salivary EBV DNA level in patients at an advanced overall stage appeared to be higher than in patients at an early stage.