Poly(3-hydroxybutyrate)-ethyl cellulose based bio-composites with novel characteristics for infection free wound healing application

A series of bio-composites including poly3-hydroxybutyrate [P(3HB)] grafted ethyl cellulose (EC) stated as P(3HB)-EC were successfully synthesised. Furthermore, natural phenols e.g., p-4-hydroxybenzoic acid (HBA) and ferulic acid (FA) were grafted onto the newly developed P(3HB)-EC-based bio-composites under laccase-assisted environment without the use of additional initiators or crosslinking agents. The phenol grafted bio-composites were critically evaluated for their antibacterial and biocompatibility features as well as their degradability in soil. In particular, the results of the antibacterial evaluation for the newly developed bio-composites indicated that 20HBA-g-P(3HB)-EC and 15FA-g-P(3HB)-EC bio-composites exerted strong bactericidal and bacteriostatic activity against Gram- E. coli NTCT 10418 as compared to the Gram+ B. subtilis NCTC 3610. This study shows further that at various phenolic concentrations the newly synthesised bio-composites remained cytocompatible with human keratinocyte-like HaCaT skin cells, as 100% cell viability was recorded, in vitro. As for the degradation, an increase in the degradation rate was recorded during the soil burial analyses over a period of 42 days. These findings suggest that the reported bio-composites have great potential for use in wound healing; covering the affected skin area which may favour tissue repair over shorter periods

Development of bio-composites with novel characteristics: Evaluation of phenol-induced antibacterial, biocompatible and biodegradable behaviours

This paper describes a laccase-assisted grafting of gallic acid (GA) and thymol (T) as functional entities onto the previously developed P(3HB)-g-EC composite. GA-g-P(3HB)-g-EC and T-g-P(3HB)-g-EC bio-composites were prepared by laccase-assisted free radical-induced graft polymerisation of GA and T onto the P(3HB)-g-EC based composite using surface dipping and incorporation technique. The results of the antibacterial evaluation for the prepared composites indicated that 15GA-g-P(3HB)-g-EC, 15T-g-P(3HB)-g-EC and 20T-g-P(3HB)-g-EC composites possessed the strongest bacteriostatic and bactericidal activities against Gram-positive B. subtilis NCTC 3610 and S. aureus NCTC 6571 and Gram-negative E. coli NTCT 10418 and P. aeruginosa NCTC 10662 strains. In this study, we have also tested GA-g-P(3HB)-g-EC and T-g-P(3HB)-g-EC bio-composites for their ability to support and maintain multilineage differentiation of human keratinocyte-like (HaCaT) skin cells in-vitro. From the cytotoxicity results, the tested composites showed 100% viability and did not induce any adverse effect on a HaCaT’s morphology. Finally, in soil burial evaluation, a progressive increase in the degradation rate of GA-g-P(3HB)-g-EC and T-g-P(3HB)-g-EC bio-composites was recorded with the passage of time up to 6 weeks. In summary, our current findings suggest that GA-g-P(3HB)-g-EC and T-g-P(3HB)-g-EC bio-composites are promising candidates for biomedical type applications such as skin regeneration, multiphasic tissue engineering and/or medical implants

Novel anti-inflammatory and chondroprotective effects of the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and human melanocortin MC3 receptor agonist PG-990 on lipopolysaccharide activated chondrocytes

Human melanocortin MC1 and MC3 receptors expressed on C-20/A4 chondrocytes exhibit chondroprotective and anti-inflammatory effects when activated by melanocortin peptides. Nearly 9 million people in the UK suffer from osteoarthritis, and bacterial infections play a role in its development. Here, we evaluate the effect of a panel of melanocortin peptides with different selectivity for human melanocortin MC1 (alpha-MSH, BMS-470539 dihydrochloride) and MC3 receptors ([DTrp8]-g-MSH, PG-990) and C-terminal peptide alpha-MSH11-13(KPV), on inhibiting LPS-induced chondrocyte death, pro-inflammatory mediators and induction of anti-inflammatory proteins. C-20/A4 chondrocytes were treated with a panel of melanocortin peptides prophylactically and therapeutically in presence of LPS (0.1 ug/ml). The chondroprotective properties of these peptides determined by cell viability assay, RT-PCR, ELISA for detection of changes in inflammatory markers (IL-6, IL-8 and MMP-1, -3 and -13) and western blotting for expression of the anti-inflammatory protein heme-oxygenase-1. C-20/A4 expressed human melanocortin MC1 and MC3 receptors and melanocortin peptides elevated cAMP. LPS stimulation caused a reduction in C-20/A4 viability, attenuated by the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride, and MC3 receptor agonists PG-990 and [DTrp8]-g-MSH. Prophylactic and therapeutic regimes of [DTrp8]-g-MSH significantly inhibited LPS-induced modulation of cartilage-damaging IL-6, IL-8, MMPs -1,-3 and -13 mediators both prophylactically and therapeutically, whilst human melanocortin MC1 and MC3 receptor agonists promoted an increase in HO-1 production. In the presence of LPS, activation of human melanocortin MC1 and MC3 receptors provided potent chondroprotection, upregulation of anti-inflammatory proteins and downregulation of inflammatory and proteolytic mediators involved in cartilage degradation, suggesting a new avenue for osteoarthritis treatment

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 μM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5′-dithiobis 2-nitrobenzoic acid (DTNB) (100 μM) or inhibiting PDI with bacitracin (5 mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 μM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 μM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain

Autoantibodies to neutrophil granule proteins: pathogenic potential in vasculitis?

The discovery of circulating autoantibodies to neutrophil granule proteins (ANCA), which are present in a high proportion of patients with vasculitis, has revitalised both clinical and experimental research in this area. Although their antigen specificities and strong association with vasculitic diseases have now been established, the role of ANCA in the disease process is uncertain. This review is a brief outline of some of the clinical associations of ANCA, and describes interesting new avenues being taken in recent research which may provide an insight into how ANCA may contribute to vascular damage in patients with vasculitis

The melanocortin receptor antagonist SHU9119 inhibits the anti-inflammatory/pro-resolving properties of a-MSH and D[TRP8]-y-MSH in lipopolysaccharide stimulated chondrocytes

Introduction: Infectious arthritis occurs when E.coli or other microorganisms lead to joint infection and increases in pro-inflammatory cytokines. Investigating how the body naturally produced anti-inflammatory proteins inhibit these pathways, may lead to the development of new therapies for these pathologies (1). The melanocortin agonists α-MSH and D[Trp8]-γ-MSH (2) display an important role in inhibiting inflammatory mediators and inducing pro-resolving anti-inflammatory pathways, in models of acute and chronic inflammation (1). The anti-inflammatory effects of these peptides are via a family of G-protein coupled receptors, of which five melanocortin receptors (MC) have been identified, with both MC1 and MC3 being promising candidates for modulation of the host inflammatory response in arthritic pathologies (3). This study aims to determine whether the MC3/4 antagonist SHU9119 modulates the anti-inflammatory, pro-resolving effects of α-MSH and D[Trp8]-γ-MSH in lipopolysaccharide (LPS) stimulated chondrocytes. Methods: Human C20/A4 cell-line chondrocytes were plated at 1x106 cells/well in 24-well plates and were pre-treated with the pan-melanocortin agonist α-MSH (3µg/ml) (Sigma–Aldrich Inc. Poole, UK), and the MC3 agonist D[Trp8]-γ-MSH (3 µg/ml) (Phoenix Pharmaceuticals, Karlsrhue, Germany) (all dissolved in PBS) for 30mins prior to 0.1µg/ml of LPS (E.coli;111.60) (Sigma–Aldrich Inc. Poole, UK) stimulation for 6h. In separate experiments the cells were pre-treated with the MC3/4 antagonist SHU9119 (10µg/ml) (Bachem, Bubendorf, Switzerland) 1h prior to LPS stimulation (Time 0h). Cells were harvested to determine the anti-inflammatory protein heme-oxygenase 1 (HO-1) expression by western blot, whilst cell-free supernatants were collected and analysed for IL-6 and IL-8 release by ELISA. Data are expressed as Mean ± SD of n=4 determination in quadruplicate. *P<0.05 vs. control. Results: Cytokine analysis: LPS 0.1ug/ml caused a maximal release of IL-6 and IL-8 with 112.3±6.1 and 314.7±1.9 pg/ml respectively (P<0.05 vs. control). Pre-treatment of cells with α-MSH and D[Trp8]-γ-MSH caused a significant reduction in IL-6 (61% and 70% respectively) and IL-8 (71% and 59% respectively) following LPS stimulation. The antagonist SHU9119 completely attenuated the effect of α-MSH on IL-6 and IL-8 release, whilst significantly reducing the effect of D[Trp8]-γ-MSH for both cytokines. HO-1 expression: LPS caused a 30% (0.70 fold) reduction in HO-1 protein expression compared to control, whilst pre-treatment of cells with α-MSH, and D[Trp8]-γ-MSH caused a significant increase in HO-1 expression with a 1.25 and 1.57 fold increase respectively. Pre-treatment with the antagonist SHU9119 inhibited both α-MSH and D[Trp8]-γ-MSH induced HO-1 expression. Conclusion: In conclusion α-MSH and D[Trp8]-γ-MSH significantly inhibit proinflammatory cytokine release whilst inducing the anti-inflammatory protein HO-1. These effects are attenuated in the presence of the MC3/4 antagonist SHU9119, thus suggesting a potential role for the MC3 receptor in mediating their anti-inflammatory effects in this model