Self-esterified hyaluronan hydrogels: Advancements in the production with positive implications in tissue healing.

Hyaluronan-(HA) short half-life in vivo limits its benefits in tissue repair. Self-esterified-HA is of great interest because it progressively releases HA, promoting tissue-regeneration longer than the unmodified-polymer. Here, the 1-ethyl-3-(3-diethylaminopropyl)carbodiimide(EDC)-hydroxybenzotriazole(HOBt) carboxyl-activating-system was evaluated for self-esterifying HA in the solid state. The aim was to propose an alternative to the time-consuming, conventional reaction of quaternary-ammonium-salts of HA with hydrophobic activating-systems in organic media, and to the EDC-mediated reaction, limited by by-product formation. Additionally, we aimed to obtain derivatives releasing defined molecular-weight(MW)-HA that would be valuable for tissue renewal. A 250 kDa-HA(powder/sponge) was reacted with increasing EDC/HOBt amounts. HA-modification was investigated through Size-Exclusion-Chromatography-Triple-Detector-Array-analyses, FT-IR/1H NMR and the products(XHAs) extensively characterized. Compared to conventional protocols, the set procedure is more efficient, avoids side-reactions, allows for an easier processing to diverse clinically-usable 3D-forms, leads to products gradually releasing HA under physiological conditions with the possibility to tune the MW of the biopolymer-released. Finally, the XHAs exhibit sound stability to Bovine-Testicular-Hyaluronidase, hydration/mechanical properties suitable for wound-dressings, with improvements over available matrices, and prompt in vitro wound-regeneration, comparably to linear-HA. To the best of our knowledge, the procedure is the first valid alternative to conventional protocols for HA self-esterification with advances in the process itself and in product performance.

Exploiting Potential Biotechnological Applications of Poly-γ-glutamic Acid Low Molecular Weight Fractions Obtained by Membrane-Based Ultra-Filtration.

Since the potentialities of applications of low molecular weight poly-γ-glutamic acid (γ-PGA) chains have been so far only partially explored, the separation of diverse molecular families of them, as well as their characterization for potential bioactivity and ability to form films, were investigated. Two different approaches based on organic solvent precipitation or on ultra- and nano-filtration membrane-based purification of inexpensive commercial material were employed to obtain size-specific γ-PGA fractions, further characterized by size exclusion chromatography equipped with a triple detector array and by ultra-high-performance liquid chromatography to assess their average molecular weight and their concentration. The γ-PGA low molecular weight fractions, purified by ultra-filtration, have been shown both to counteract the desiccation and the oxidative stress of keratinocyte monolayers. In addition, they were exploited to prepare novel hydrocolloid films by both solvent casting and thermal compression, in the presence of different concentrations of glycerol used as plasticizer. These biomaterials were characterized for their hydrophilicity, thermal and mechanical properties. The hot compression led to the attainment of less resistant but more extensible films. However, in all cases, an increase in elongation at break as a function of the glycerol content was observed. Besides, the thermal analyses of hot compressed materials demonstrated that thermal stability was increased with higher γ-PGA distribution po-lymer fractions. The obtained biomaterials might be potentially useful for applications in cosmetics and as vehicle of active molecules in the pharmaceutical field.

Label-Free Quantitative Proteomics to Explore the Action Mechanism of the Pharmaceutical-Grade Triticum vulgare Extract in Speeding Up Keratinocyte Healing.

Plant extracts have shown beneficial properties in terms of skin repair, promoting wound healing through a plethora of mechanisms. In particular, the poly-/oligosaccharidic aqueous extract of Triticum vulgare (TVE), as well as TVE-based products, shows interesting biological assets, hastening wound repair. Indeed, TVE acts in the treatment of tissue regeneration mainly on decubitus and venous leg ulcers. Moreover, on scratched monolayers, TVE prompts HaCat cell migration, correctly modulating the expression of metalloproteases toward a physiological matrix remodeling. Here, using the same HaCat-based in vitro scratch model, the TVE effect has been investigated thanks to an LFQ proteomic analysis of HaCat secretomes and immunoblotting. Indeed, the unbiased TVE effect on secreted proteins has not yet been fully understood, and it could be helpful to obtain a comprehensive picture of its bio-pharmacological profile. It has emerged that TVE treatment induces significant up-regulation of several proteins in the secretome (153 to be exact) whereas only a few were down-regulated (72 to be exact). Interestingly, many of the up-regulated proteins are implicated in promoting wound-healing-related processes, such as modulating cell-cell interaction and communication, cell proliferation and differentiation, and prompting cell adhesion and migration.

Chondroitin Sulfate in USA Dietary Supplements in Comparison to Pharma Grade Products: Analytical Fingerprint and Potential Anti-Inflammatory Effect on Human Osteoartritic Chondrocytes and Synoviocytes.

The biological activity of chondroitin sulfate (CS) and glucosamine (GlcN) food supplements (FS), sold in USA against osteoarthritis, might depend on the effective CS and GlcN contents and on the CS structural characteristics. In this paper three USA FS were compared to two pharmaceutical products (Ph). Analyses performed by HPAE-PAD, by HPCE and by SEC-TDA revealed that the CS and GlcN titers were up to -68.8% lower than the contents declared on the labels and that CS of mixed animal origin and variable molecular weights was present together with undesired keratan sulfate. Simulated gastric and intestinal digestions were performed in vitro to evaluate the real CS amount that may reach the gut as biopolymer. Chondrocytes and synoviocytes primary cells derived from human pathological joints were used to assess: cell viability, modulation of the NF-κB, quantification of cartilage oligomeric matrix protein (COMP-2), hyaluronate synthase enzyme (HAS-1), pentraxin (PTX-3) and the secreted IL-6 and IL-8 to assess inflammation. Of the three FS tested only one (US FS1) enhanced chondrocytes viability, while all of them supported synoviocytes growth. Although US FS1 proved to be less effective than Ph as it reduced NF-kB, it could not down-regulate COMP-2; HAS-1 was up-regulated but with a lower efficacy. Inflammatory cytokines were markedly reduced by Ph while a slight decrease was only found for US-FS1.

Comparative Analyses of Pharmaceuticals or Food Supplements Containing Chondroitin Sulfate: Are Their Bioactivities Equivalent?

INTRODUCTION: Oral supplementation of chondroitin sulfate (CS) and glucosamine (GlcN), symptomatic slow-acting molecules, is recommended by European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis and Musculoskeletal Diseases (ESCEO) and other European Union (EU) guidelines for the restoration of the articular cartilage surface in patients affected by osteoarthritis (OA). They are commercialized as pharmaceutical grade products and as food supplements in combination with plant extracts hyaluronic acid, methylsulfonylmethane, and other components. Food supplements do not need to undergo the strict regulatory controls of pharmaceutical grade products; thus, composition and contaminants that could be present may not be evidenced before commercialization and these uncertainties may give rise to concerns about the bioactivity of these formulations. METHODS: In this paper 10 different food supplements (FS) from diverse European countries were analyzed in comparison with two pharmaceutical grade products (Ph) using updated analytical approaches and biochemical cell-based assays. The purity, the titer, and the origin of CS in Ph and FS samples were initially assessed in order to successively compare the biological function. Both food supplements and pharmaceutical formulations were tested in vitro, using the same final CS concentration, on primary chondrocytes and synoviocytes in terms of (i) cell viability, (ii) activation of the NF-κB-mediated inflammation pathway, (iii) cartilage oligomeric matrix protein (COMP-2), IL-6, and IL-8 production. RESULTS: All the FS presented a certain insoluble fraction; the CS and the GlcN contents were lower than the declared ones in 9/10 and 8/10 samples, respectively. All FS contained keratan sulfate (KS) at up to 50% of the total glycosaminoglycan amount declared on the label. Primary cells treated with the samples diluted to present the same CS concentration in the medium showed cytotoxicity in 7/10 FS while Ph preserved viability and reduced NF-κB, COMP-2, and secreted inflammatory cytokines. CONCLUSION: Among all samples tested, the pharmaceutical grade products demonstrated effective modulation of biomarkers counteracting the inflammation status and improving viability and the physiological condition of OA human primary chondrocyte and synoviocyte cells. In contrast to that, most FS were cytotoxic at the tested concentrations, and only 3/10 of them showed similarities to Ph sample behavior in vitro. FUNDING: This work was partially supported by PON01_1226 NUTRAFAST, MIUR Ministero dell'Università e della Ricerca Scientifica. Bioteknet financed two short-term grants for graduate technicians. The journal's Rapid Service and Open Access fees were funded by IBSA CH.

Molecular weight determination of heparosan- and chondroitin-like capsular polysaccharides: figuring out differences between wild -type and engineered Escherichia coli strains.

Heparin and chondroitin sulfate are used as anti-thrombic and anti-osteoarthritis drugs, respectively, but their pharmacological actions depend on their structural characteristics such as their sulfation grade and their molecular weight. In the last years, new fermentation-based biotechnological approaches have tried to obtain heparin and chondroitin sulfate starting from the heparosan and chondroitin-like capsular polysaccharides produced by Escherichia coli K5 and K4. The study of the microbial capsular polysaccharide molecular weight is critical to obtain nature-like or structural tailor cut glycosaminoglycan homologues. However, so far, it has been scarcely investigated. In this paper, for the first time, a new protocol was set up to determine the molecular weights of the capsular polysaccharides of three wild-type and three engineered E. coli K5 and K4 strains. The protocol includes a small-scale downstream train to purify the intact polysaccharides, directly from the fermentation broth supernatants, by using ultrafiltration membranes and anion exchange chromatography, and it couples size exclusion chromatography analyses with triple detector array. In the purification high recovery (> 85.0%) and the removal of the main contaminant, the lipopolysaccharide, were obtained. The averaged molecular weights of the wild-type capsular polysaccharides ranged from 51.3 to 90.9 kDa, while the engineered strains produced polysaccharides with higher molecular weights, ranging from 68.4 to 130.6 kDa, but with similar polydispersity values between 1.1 and 1.5.