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Cardiac tissue engineering (CTE) is currently a prime focus of research due to an enormous clinical need. In this work, a novel functional material, Poly(3-hydroxyoctanoate), P(3HO), a medium chain length polyhydroxyalkanoate (PHA), produced using bacterial fermentation, was studied as a new potential material for CTE. Engineered constructs with improved mechanical properties, crucial for supporting the organ during new tissue regeneration, and enhanced surface topography, to allow efficient cell adhesion and proliferation, were fabricated. Our results showed that the mechanical properties of the final patches were close to that of cardiac muscle. Biocompatibility of the P(3HO) neat patches, assessed using Neonatal ventricular rat myocytes (NVRM), showed that the polymer was as good as collagen in terms of cell viability, proliferation and adhesion. Enhanced cell adhesion and proliferation properties were observed when porous and fibrous structures were incorporated to the patches. Also, no deleterious effect was observed on the adults cardiomyocytes’ contraction when cardiomyocytes were seeded on the P(3HO) patches. Hence, P(3HO) based multifunctional cardiac patches are promising constructs for efficient CTE. This work will provide a positive impact on the development of P(3HO) and other PHAs as a novel new family of biodegradable functional materials with huge potential in a range of different biomedical applications, particularly CTE, leading to further interest and exploitation of these materials.

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 (α-MSH, BMS-470539 dihydrochloride) and MC3 ([DTrp8]-γ-MSH, PG-990) receptors and C-terminal peptide α-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 μg/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]-γ-MSH. Prophylactic and therapeutic regimes of [DTrp8]-γ-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.

Bioactive glass (BG) is considered an ideal material for haemostasis as it releases Ca2+ ions upon hydration, which is required to support thrombosis. In this study the effect of the presence of the BG nanoparticles in P(3HB) microsphere films on the structural properties, thermal properties and biocompatibility of the films were studied. The nanoscaled bioactive glass with a high surface area was also tested for its in vitro haemostatic efficacy and was found to be able to successfully reduce the clot detection time. In an effort to study the effect of the roughness induced by the formation of HA on the cellular functions such as cell adhesion, cell mobility and cell differentiation, the composite films were immersed in SBF for a period of 1, 3 and 7 days. From the SEM images the surface of the P(3HB)/n-BG composite microsphere films appeared fairly uniform and smooth on day 1, however on day 3 and day 7 a rough and uneven surface was observed. The presence of HA on the composite microsphere films on day 3 and day 7 influenced the surface roughness of the films. However, when the P(3HB)/n-BG composite microspheres with enhanced surface roughness were tested for biocompatibility, reduced amount of protein adsorption and cell adhesion were observed. This study thus revealed that there is an optimal surface roughness for the P(3HB) microsphere films for increased cell adhesion, beyond which it could be deleterious for cell adhesion and differentiation.

Background:Osteoarthritis (OA) is a degenerative joint disease that affects over 250 million people worldwide [1] with treatments focussing on the symptoms rather than the cause of the pathology [2, 3]. Thus, this degenerative joint disease requires novel treatment options [3, 4].Therefore, the melanocortin system [4] could provide a novel avenue to explore given its ability to exert anti-inflammatory effects and chondroprotection [5], although the receptor subtype involved is unclear.Objectives:This study aims to assess the chondroprotective and anti-inflammatory effects of the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and the selective human MC3 receptor agonist PG-990 on S-Nitroso-N-acetyl-DL-penicillamine (SNAP) activated chondrocytes.Methods:The human chondrocytic cell-line C-20/A4 was seeded at 25.0 x 106viable cells/ml (5 μl droplet was transferred into individual wells of a 96-well plate). Micromass cultures [6] were stimulated with SNAP (1.0 mM) and after 2h treated with Dexamethasone (1.0 μM), selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride (10.0 μg/ml) or selective human melanocortin MC3 receptor agonist PG-990 (10.0 μg/ml) for 6h. Cell viability was determined by MTT assay, Caspase -3 and -7 activity determined by Caspase-Glo 3/7 apoptosis assay. Glycosaminoglycan (GAG) content determined by alcian blue staining and anti-inflammatory heme-oxygenase-1 (HO-1) protein expression was determined by western blot. Data are expressed as Mean ±S.E.M ofn=4 samples repeated in triplicate. #p≤0.05vscontrol or *p≤0.05vsstimulus.Results:Cell viability analysis showed SNAP stimulation caused a maximal cell death of 23% (#p≤0.05), Dexamethasone, BMS-470539 dihydrochloride and PG-990 inhibited cell death by 2%, 98% and 129% respectively (*p≤0.05). SNAP stimulation caused a significant increase in Caspase -3 and -7 activity, which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 8%, 5% and 19% respectively (*p≤0.05). GAG content was significantly reduced by SNAP by 29% (#p≤0.05), which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 1%, 3% and 14% respectively (*p≤0.05). SNAP also caused a significant decrease in HO-1 protein expression, which was increased by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by a 1.0-fold, 1.1-fold and 2.1-fold increase respectively (*p≤0.05).Conclusion:The selective human melanocortin MC3 receptor agonist PG-990 exhibited enhanced chondroprotection and modulation of inflammatory and tissue destructive mediators following SNAP activation compared to Dexamethasone and the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride. This suggests that melanocortin peptides display enhanced chondroprotective and anti-inflammatory effects at the MC3 receptor sub-type in this cell line.References:[1]Hunter DJ and Bierma-Zeinstra S. (2019).Lancet.393: 1745–59.[2]Can VCet al.(2020).Euro J Pharmacol. doi:https://doi.org/10.1016/j.ejphar.2020.172971.[3]Intekhab-Alam NYet al. (2013).Cell death & disease.4: 1-6.[4]Getting SJet al.(2006).Mol Pharmacol70: 1850-1855.[5]Kaneva MKet al.(2014).Biochem Pharmacol92: 336-47.[6]Greco KVet al.(2011).Biochem Pharmacol82: 1919-29.Disclosure of Interests:None declared

Purpose Articular cartilage is an avascular connective tissue present in joints which covers the ends of opposing bones providing a smooth, shock absorbing surface to promote normal joint function. Normal cartilage structure is maintained by chondrocytes, the only cell type present. Abnormal function/death of these cells, due to long- term “wear and tear” and other risk factors results in cartilage loss and osteoarthritis (OA). There is currently no cure for OA, the most common treatments simply involve the management of joint pain, or surgical treatments, which are not able to restore cartilage function. In recent years, cartilage repair has been approached with engineered scaffolds, able to support the growth of chondrocytes or progenitor cells to generate a neo-tissue, but the neo tissue generated is often fibrocartilage, mechanically inferior and less–durable than the hyaline cartilage normally found in healthy articular joints, indicating that further research is required to develop a more suitable scaffolds. The aim of this study has been to assess the properties of collagen I hydrogel 3D scaffolds, populated with the phenotypically stable chondrocytes cell line C20/A4, strengthened with solvent cast and 3D printed Polyhydroxyalkanoate (PHA) polymer. PHAs are a family of biopolyesters synthesized by bacteria with monomers of varying chain lengths (C4-C16) which can be blended to produce products with different mechanical properties. PHAs are biodegradable, non-toxic and biocompatible. It has been already established that PHA scaffolds are able to support chondrocyte growth and activity but despite that, they do not represent the natural environment for chondrocyte metabolism, collagen production and cartilage regeneration. A combination collagen hydrogel and PHA scaffold that addresses this issue would be highly desirable. Methods Polymer production and preparation PHAs were produced by Pseudomonas mendocina CH50 in batch fermentation conditions using a 15 L bioreactor with an operating fermenter volume of 10 L. with glucose as the sole carbon source under nitrogen limiting conditions. PHA film and scaffolds were either prepared using the solvent casting method incorporating large NaCl crystals (which were later dissolved out) to form a porous structure or with a 3D melt printing method. Collagen gel preparation and seeding: Type I collagen constructs were prepared with chondrocytes by mixing type I collagen polymer suspension with 10modified Eagle’s medium (MEM) and 1M NaOH solution was added dropwise, to neutralise the acidic gel mixture. This gel solution was then mixed with a cell suspension of C-20/A4 cells in Dulbecco’s MEM (DMEM)-based medium containing 1% foetal calf serum (FCS) . 1.2 ml constructs were cast in 24 well plates either on their own or with the addition of solvent cast PHA polymers or 3D printed PHA polymers. Gels were then allowed to solidify by incubation at 37 C for 5-10 minutes, overlaid with DMEM medium (1% FCS) and cultured for 1 or 7 days, at 37 C and 5% CO2. Compression Studies: Gels were subjected to compression, at 0,6KN/m2 for 5 minutes in a custom built apparatus. The displacement of gels was measured over time. Results The compression testing data demonstrates that type I collagen gels, seeded with C-20/A4 chondrocytes (control gels), exhibit changes in their mechanical properties over time, with an increased resistance and smaller displacement at Day 7 compare to Day 1. The addition of solvent cast and 3D printed scaffolds increase the mechanical resistance of the construct when compared to the populated control gels (collagen matix only) at DAY 1 and is even more evident at DAY 7. 3D printed scaffolds also show higher stiffness of the construct compared to the control and solvent cast polymer samples. Cytotoxicity testing performed with Alamar blue staining, on chondrocyte populations grown on PHA films, indicates that there are no cytotoxic effects of the polymer on cell viability at any of time points analysed over 7 days. Trypan Blue staining, after 5 minutes compression test, reveals no significant difference in viability in any of the samples containing PHA polymer (solvent cast or 3D printed) compare to control samples. Conclusions In conclusion, these studies suggest that a composite collagen gel-PHA construct provides a supportive environment for the maintenance of chondrocyte activity increasing the initial resistance of the structure to compression, whilst the embedded chondrocytes remodel the Type I collagen gel to form a more cartilage like structure. This may offer a potential application of collagen hydrogel-PHA scaffolds for clinical use in support of cartilage regeneration in small and contained osteoarthritic lesions.

Urocortin (Ucn 1), a 40 amino acid long peptide related to corticotropin releasing factor (CRF) was discovered 19 years ago, based on its sequence homology to the parent molecule. Its existence was inferred in the CNS because of anatomical and pharmacological discrepancies between CRF and its two receptor subtypes. Although originally found in the brain, where it has opposing actions to CRF and therefore confers stress-coping mechanisms, Ucn 1 has subsequently been found throughout the periphery including heart, lung, skin, and immune cells. It is now well established that this small peptide is involved in a multitude of physiological and pathophysiological processes, due to its receptor subtype distribution and promiscuity in second messenger signalling pathways. As a result of extensive studies in this field, there are now well over one thousand peer reviewed publications involving Ucn 1. In this review, we intend to highlight some of the less well known actions of Ucn 1 and in particular its role in neuronal cell protection and maintenance of the skeletal system, both by conventional methods of reviewing the literature and using bioinformatics, to highlight further associations between Ucn 1 and disease conditions. Understanding how Ucn 1 works in these tissues, will help to unravel its role in normal and pathophysiological processes. This would ultimately allow the generation of putative medical interventions for the alleviation of important diseases such as Parkinson's disease, arthritis, and osteoporosis.

International audience; We have developed novel composites by grafting caffeic acid (CA) onto the P(3HB)-EC based material and laccase from Trametes versicolor was used for grafting purposes. The resulting composites were designated as CA-g-P(3HB)EC i.e., P(3HB)-EC (control), 5CA-g-P(3HB)-EC, 10CA-g-P(3HB)-EC, 15CA-g-P(3HB)-EC and 20CA-g-P(3HB)-EC. FT-IR (Fourier-transform infrared spectroscopy) was used to examine the functional and elemental groups of the control and laccase-assisted graft composites. Evidently, 15CA-g-P(3HB)-EC composite exhibited resilient antibacterial activity against Gram-positive and Gram-negative bacterial strains. Moreover, a significant level of biocompatibility and biodegradability of the CA-g-P(3HB)-EC composites was also achieved with the human keratinocytes-like HaCaT cells and soil burial evaluation, respectively. In conclusion, the newly developed novel composites with multi characteristics could well represent the new wave of biomaterials for medical applications, and more specifically have promising future in the infection free would dressings, burn and/or skin regeneration field due to their sophisticated characteristics.

Osteoarthritis (OA) is characterised by progressive destruction of articular cartilage and chondrocyte cell death. Here, we show the expression of the endogenous peptide urocortin1 (Ucn1) and two receptor subtypes, CRF-R1 and CRF-R2, in primary human articular chondrocytes (AC) and demonstrate its role as an autocrine/paracrine pro-survival factor. This effect could only be removed using the CRF-R1 selective antagonist CP-154526, suggesting Ucn1 acts through CRF-R1 when promoting chondrocyte survival. This cell death was characterised by an increase in p53 expression, and cleavage of caspase 9 and 3. Antagonism of CRF-R1 with CP-154526 caused an accumulation of intracellular calcium (Ca2+) over time and cell death. These effects could be prevented with the non-selective cation channel blocker Gadolinium (Gd3+). Therefore, opening of a non-selective cation channel causes cell death and Ucn1 maintains this channel in a closed conformation. This channel was identified to be the mechanosensitive channel Piezo1. We go on to determine that this channel inhibition by Ucn1 is mediated initially by an increase in cyclic adenosine monophosphate (cAMP) and a subsequent inactivation of phospholipase A2 (PLA2), whose metabolites are known to modulate ion channels. Knowledge of these novel pathways may present opportunities for interventions that could abrogate the progression of OA.

Novel Poly(3-hydroxybutyrate)/Poly(3-hydroxyoctanoate) blends were developed with varying amounts of Poly(3-hydroxyoctanoate), P(3HO) and Poly(3-hydroxybutyrate), P(3HB) for their potential use in various medical applications. These blend films exhibited higher tensile strength and Young’s modulus values compared to neat P(3HO). The overall protein adsorption and % cell viability was also found to be significantly higher in the blend films than the neat P(3HO) film. Hydrolytic degradation was faster in the blend films and the degradation rate could potentially be tailored to achieve the optimum rate required for a particular medical application. Hence, these novel blends were found to be highly biocompatible with surface, mechanical and thermal properties suitable for a range of potential medical applications, a great step forward in the area of medical materials.

Poly(3-hydroxyoctanoate)/poly(3-hydroxybutyrate), P(3HO)/P(3HB), blend films loaded with aspirin were prepared, and the influence of aspirin loading on the surface properties, mechanical, thermal and degradation properties were investigated. Scanning electron microscopy images revealed that the addition of aspirin introduced a new topography on the surface of the blend films. Aspirin contributed to the increase in the hydrophilic nature of the blend films compared with the unloaded blend films. This was complemented by a considerable increase in the total protein adsorption in the aspirin-loaded blend films. The percentage cell viability was higher in the aspirin-loaded blend films compared with the unloaded blend films. There was a decrease in the tensile strength and the Young’s modulus with the addition of the aspirin. However, the percentage elongation at break, a measure of elasticity, was higher in the aspirin-loaded films, indicating an increase in their flexibility compared with the unloaded blend films. There was a decrease in the melting temperature (Tm), glass transition temperature (Tg) and the crystallization temperature (Tc) due to the decrease in the crystallinity of the aspirin-loaded blend films in comparison with the unloaded blend films. Finally, controlled release of aspirin was observed without any burst release, and 96·6% release was achieved within 25 d, ideal for the development of biodegradable drug-eluting stents.

A novel nanocomposite material which combines the biocompatible, elastomeric, natural, biodegradable homopolymer, poly(3-hydroxyoctanoate), P(3HO), with haemostatic and antibacterial bioactive glass nanoparticles (n-BG), were developed as a matrix for skin related applications. P(3HO) is a unique member of the family of natural polyhydroxyalkanoate biopolymers. The P(3HO)/n-BG composite films were fabricated using the solvent casting method. Microstructural studies revealed n-BG particles both embedded in the matrix and deposited on the surface which introduced nanotopography and increased its hydrophilicity. The composite exhibited an increase in the Young's modulus when compared to the control, yet maintained flexible elastomeric properties. These changes in the surface topography and chemistry of the composite system led to an increase of protein adsorption and cytocompatibility for the seeded human keratinocyte cell line (HaCaT). The results from this study demonstrated that the fabricated P(3HO)/n-BG composite system is a promising novel matrix material with potential applications in skin tissue engineering and wound healing.

BACKGROUND AND PURPOSE: Melanocortin MC1 and MC3 receptors, mediate the anti-inflammatory effects of melanocortin peptides. Targeting these receptors could therefore lead to development of novel anti-inflammatory therapeutic agents. We investigated the expression of MC1 and MC3 receptors on chondrocytes and the role of α-melanocyte-stimulating hormone (α-MSH) and the selective MC3 receptor agonist, [DTRP8]-γ-MSH, in modulating production of inflammatory cytokines, tissue-destructive proteins and induction of apoptotic pathway(s) in the human chondrocytic C-20/A4 cells. EXPERIMENTAL APPROACH: Effects of α-MSH, [DTRP8]-γ-MSH alone or in the presence of the MC3/4 receptor antagonist, SHU9119, on TNF-α induced release of pro-inflammatory cytokines, MMPs, apoptotic pathway(s) and cell death in C-20/A4 chondrocytes were investigated, along with their effect on the release of the anti-inflammatory cytokine IL-10. KEY RESULTS: C-20/A4 chondrocytes expressed functionally active MC1,3 receptors. α-MSH and [DTRP8]-γ-MSH treatment, for 30 min before TNF-α stimulation, provided a time-and-bell-shaped concentration-dependent decrease in pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) release and increased release of the chondroprotective and anti-inflammatory cytokine, IL-10, whilst decreasing expression of MMP1, MMP3, MMP13 genes.α-MSH and [DTRP8]-γ-MSH treatment also inhibited TNF-α-induced caspase-3/7 activation and chondrocyte death. The effects of [DTRP8]-γ-MSH, but not α-MSH, were abolished by the MC3/4 receptor antagonist, SHU9119. CONCLUSION AND IMPLICATIONS: Activation of MC1/MC3 receptors in C-20/A4 chondrocytes down-regulated production of pro-inflammatory cytokines and cartilage-destroying proteinases, inhibited initiation of apoptotic pathways and promoted release of chondroprotective and anti-inflammatory cytokines. Developing small molecule agonists to MC1/MC3 receptors could be a viable approach for developing chondroprotective and anti-inflammatory therapies in rheumatoid and osteoarthritis.

Purpose: Single injurious mechanical impact to joints, as seen in sports injury, is known to be associated with a high risk of later development of osteoarthritis (OA). Irreversible chondrocyte death is initiated following impact and results in progressive breakdown of articular cartilage. Promotion of chondrocyte survival is therefore a necessary target in the prevention of development of further OA. Urocortin1 (Ucn) has recently been shown to have a pro-survival effect on chondrocytes in both the presence and absence of pro-apoptotic stimuli. The aim of this study was to investigate the role of Ucn in response to mechanical impact. Methods: Full thickness porcine cartilage explants from lateral and medial femoral condyles were impacted with a 500g weight using a custom-made drop tower device. Impacted explants were treated with human Ucn (10-7M, 30 minutes prior to or following impact), non-selective cation channel blocker Gadolinium (500μM, 30 minutes following impact), or mechanosensitive ion channel blocker GsMTx4 (40μM, 2 hours prior to impact) and cultured for 72hrs. Cell viability was assessed with a CMFDA:PI live dead assay, using both macro confocal microscopy, as a means of avoiding bias, and high magnification confocal microscopy techniques. Lactate dehydrogenase (LDH) assays were conducted at 72hrs to provide a quantitative read out of cell death for each explant. Calcium influx was assessed 1hr post impact by Fluo-4 AM staining and visualised by multiphoton microscopy. Results: Porcine explants were impacted from a range of heights (10-70mm) in order to establish the correct energy of impact for inducing cell death without excess damage and fracture of the cartilage explant. Cell death increased in a dose dependent manner with height. 50mm was chosen as optimal height for all further experiments. Pre-treatment of explants with Ucn had a significant increase on cell viability when compared to impacted alone, as seen by quantification of the CMFDA:PI ratio on macro confocal (p=0.0042) and by high magnification confocal microscopy (p=0.0015). This increase in cell viability was confirmed by LDH assay (p=0.0063). Post-impact treatment of the explants with Ucn also resulted in a significant increase in cell survival, seen in both CMFDA:PI ratio (p=0.0009) and LDH assay (p=0.0053). We have previously found in primary human chondrocytes that Ucn exerts its protective effect on chondrocytes by the inhibition of excessive calcium influx. Calcium influx was therefore visualised in impacted explants treated pre- and post-impact with Ucn. A significant decrease in the levels of calcium staining was seen in both of these conditions when compared to explants impacted in the absence of Ucn (p=0.0073, p=0.05). In vitro work also highlighted a role for the mechanosensitive ion channel Piezo1 in cell death associated with the absence of Ucn. Explants were therefore treated with both Gadolinium and GsMTx4 post-impact and cell viability assessed by multiphoton microscopy. An increase in cell viability was observed in both conditions, comparable to those seen with addition of Ucn. Conclusions: A novel role for Ucn in the protection of articular chondrocytes maintained in cartilage explants has been shown and validated by two microscopy techniques and LDH assay. Ucn has also been shown to reduce calcium influx in impacted explants at 60 minutes post impact, and increased cell viability was observed by blocking mechanosensitive ion channels. These data suggest the importance of regulation of calcium influx in Ucn-mediated cell protection. The pro-survival effect of Ucn was seen both pre- and post-impact, highlighting the potential prophylactic and therapeutic use of Ucn for treating sudden injury to cartilage, and subsequent progression to post-traumatic OA.

The homopolymer poly(3-hydroxyoctanoate), P(3HO) produced from Pseudomonas mendocina using octanoate as carbon feed was studied as a potential biomaterial for soft tissue engineering i.e. as cardiac patch and as matrices for skin tissue engineering. The polymer was fabricated into neat solvent cast films of 5 and 10 wt %. Microstructural studies revealed the films as having smooth surface topography with a root mean square (RMS) value of 0.238 µm. The films also possessed moderate hydrophilicity when compared to other monomers of the PHA family. Stress-strain curves of the films obtained was typical of that of elastomeric polymers. This elastomeric and flexible nature of the films makes them promising candidates for the proposed applications. Biocompatibility studies using the HaCaT keratinocyte cell line showed that the films were able to support the attachment, differentiation and maturation of the HaCaT cells. In vitro degradation studies over a period of 4 months showed that the water absorption and weight loss increased progressively with time for the films. The films underwent hydrolytic degradation initiated on the surface and also showed an ageing effect.

Tetracycline, an antibiotic used against a broad range of Gram positive and Gram negative bacteria was encapsulated in microspheres made of poly(3-hydroxybutyric acid) P(3HB), a microbial biodegradable polymer isolated from Bacillus cereus SPV. The drug loaded microspheres were prepared using an oil emulsion technique and compressed uniaxially to produce films. Although the same fabrication conditions were used for preparing the drug loaded and unloaded microspheres, the presence of the drug changed the surface morphology and roughness of the films. The surface morphology of the drug loaded films appeared uneven and coarser and the roughness, with an average root mean square value of 5.89 µm, was significantly higher than that of the unloaded film. The in vitro biocompatibility of the films was investigated using a human keratinocyte cell line (HaCaT) by comparing cell viability on the films to that on conventional tissue culture plastics. Both films appear to support cell growth but cell attachment and percentage cell viability were greater on the drug loaded films (32% of control) compared to the unloaded film (10% of control), possibly as a result of the non-uniform surface morphology and increased roughness of the drug loaded film. Thus, the above results illustrate that the drug loaded films, in addition to being a suitable matrix for drug delivery, represent an improved substrate for keratinocyte cell attachment.

Arthritic pathologies are a major cause of morbidity within the western world, with rheumatoid arthritis affecting approximately 1% of adults. This review highlights the therapeutic potential of naturally occurring hormones and their peptides, in both arthritic models of disease and patients. The arthritides represent a group of closely related pathologies in which cytokines, joint destruction, and leukocytes play a causal role. Here we discuss the role of naturally occurring pro-opiomelanocortin (POMC)-derived melanocortin peptides (e.g., alpha melanocyte stimulating hormone [a-MSH]) and synthetic derivatives in these diseases. Melanocortins exhibit their biological efficacy by modulating proinflammatory cytokines and subsequent leukocyte extravasation. Their biological effects are mediated via seven transmembrane G-protein-coupled receptors, of which five have been cloned, identified, and termed MC1to MC5. Adrenocorticotrophic hormone represents the parent molecule of the melanocortins; the first 13 amino acids of which (termed a-MSH) have been shown to be the most pharmacologically active region of the parent hormone. The melanocortin peptides have been shown to display potent anti-inflammatory effects in both animal models of disease and patients. The potential anti-inflammatory role for endogenous peptides in arthritic pathologies is in its infancy. The ability to inhibit leukocyte migration, release of cytokines, and induction of anti-inflammatory proteins appears to play an important role in affording protection in arthritic injury, and thus may lead to potential therapeutic targets.

Introduction Mechanical injury can greatly influence articular cartilage, propagating inflammation, cell injury and death – risk factors for the development of osteoarthritis. Melanocortin peptides and their receptors mediate anti-inflammatory and pro-resolving mechanisms in chondrocytes. This study aimed to investigate the potential chondroprotective properties of α-MSH and [DTRP8]-γ-MSH in mechanically injured cartilage explants, their ability to inhibit pro-inflammatory and stimulate anti-inflammatory cytokines in in situ and in freshly isolated articular chondrocytes. Methods The effect of melanocortins on in situ chondrocyte viability was investigated using confocal laser scanning microscopy of bovine articular cartilage explants, subjected to a single blunt impact (1.14 N, 6.47 kPa) delivered by a drop tower. Chondroprotective effects of α-MSH, [DTRP8]-γ-MSH and dexamethasone on cytokine release by TNF-α-activated freshly isolated articular chondrocytes/mechanically injured cartilage explants were investigated by ELISA. Results A single impact to cartilage caused discreet areas of chondrocyte death, accompanied by pro-inflammatory cytokine release; both parameters were modulated by α-MSH, [DTRP8]-γ-MSH and dexamethasone. Melanocortin pre-treatment of TNF-α-stimulated freshly isolated chondrocytes resulted in a bell-shaped inhibition in IL-1β, IL-6 and IL-8, and elevation of IL-10 production. The MC3/4 antagonist, SHU9119, abrogated the effect of [DTRP8]-γ-MSH but not α-MSH on cytokine release. Conclusion Melanocortin peptide pre-treatment prevented chondrocyte death following mechanical impact to cartilage and led to a marked reduction of pro-inflammatory cytokines, whilst prompting the production of anti-inflammatory/pro-resolving cytokine IL-10. Development of small molecule agonists towards melanocortin receptors could thus be a viable approach for preventing chondrocyte inflammation and death within cartilage and represent an alternative approach for the treatment of osteoarthritis.

The potential neuroprotective action of the corticotrophin-releasing factor-related peptide urocortin (UCN) was investigated in the rat 6-hydroxydopamine (6-OHDA) and lipopolysaccharide (LPS) paradigms of Parkinson's disease. UCN (20 fmol) was either given at the same time as (T = 0) or 7 days after (T = +7) intracerebral 6-OHDA or LPS injection. At 14 days after 6-OHDA or LPS injection, circling behaviour was measured following apomorphine challenge. Circling was significantly lower in rats given UCN at either T = 0 or T = +7 compared with animals given 6-OHDA or LPS and vehicle. Sham-treated rats showed no circling. Consistent with these observations, striatal dopamine concentrations were markedly higher in 6-OHDA/LPS + UCN rats vs. 6-OHDA/LPS + vehicle groups. Additionally, L-dihydroxyphenylalanine production by tyrosine hydroxylase was greatly reduced in the striata of 6-OHDA/LPS + vehicle rats, whereas this was not the case in rats coadministered UCN. Finally, the numbers of tyrosine hydroxylase-positive cells recorded in the substantia nigra of 6-OHDA/LPS + vehicle-treated animals were markedly lower than those of sham-operated or 6-OHDA/LPS + UCN rats. Critically, UCN was effective in reversing lesion-induced deficits when given either at the same time as or 7 days after the neurotoxic insult. To our knowledge, this is the first time that such an effect has been demonstrated in vivo. The apparent ability of UCN to arrest the progression of or even reverse nigral lesions once established suggests that pharmacological manipulation of this system could have substantial therapeutic utility.

Streptodornase, a streptococcal deoxyribonuclease present as an active constituent of the medicament Varidase®, has been shown to exhibit no activity against RNA and digests native DNA more rapidly than single-stranded DNA. The pH range of streptodornase activity has also been established and the enzyme has been shown to exhibit DNase activity over a wide pH range (5.0–9.0) exhibiting maximal activity at pH 7.8. The effects of temperature on streptodornase activity have also been investigated under a variety of conditions showing the enzyme to be active over a range of 10–60 °C. The data from these temperature studies would suggest the possible existence of semi-denatured ‘meta-stable’ conformations for the streptodornase molecule with reduced levels of DNase activity. The effect of variable substrate concentrations has also been studied to enable the determination of the kinetic constants for this enzyme. The lack of RNAse activity by streptodornase would support data showing a low immunogenic response to Varidase® treatment.

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 Bacillus subtilis NCTC 3610 and Staphylococcus aureus NCTC 6571 and Gram-negative Escherichia coli NTCT 10418 and Pseudomonas 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.

Recently, the development of highly inspired biomaterials with multi-functional characteristics has gained considerable attention, especially in biomedical, and other health-related areas of the modern world. It is well-known that the lack of antibacterial potential has significantly limited biomaterials for many challenging applications such as infection free wound healing and/or tissue engineering etc. In this perspective, herein, a series of novel bio-composites with natural phenols as functional entities and keratin-EC as a base material were synthesised by laccase-assisted grafting. Subsequently, the resulting composites were removed from their respective casting surfaces, critically evaluated for their antibacterial and biocompatibility features and information is also given on their soil burial degradation profile. In-situ synthesised phenol-g-keratin-EC bio-composites possess strong anti-bacterial activity against Gram-positive and Gram-negative bacterial strains i.e., B. subtilis NCTC 3610, P. aeruginosa NCTC 10662, E. coli NTCT 10418 and S. aureus NCTC 6571. More specifically, 10HBA-g-keratin-EC and 20T-g-keratin-EC composites were 100% resistant to colonisation against all of the aforementioned bacterial strains, whereas, 15CA-g-keratin-EC and 15GA-g-keratin-EC showed almost negligible colonisation up to a variable extent. Moreover, at various phenolic concentrations used, the newly synthesised composites remained cytocompatible with human keratinocyte-like HaCaT, as an obvious cell ingrowth tendency was observed and indicated by the neutral red dye uptake assay. From the degradation point of view, an increase in the degradation rate was recorded during their soil burial analyses. Our investigations could encourage greater utilisation of natural materials to develop bio-composites with novel and sophisticated characteristics for potential applications.

The activity of streptodornase, a deoxyribonuclease active protein component of the medicament Varidase® has been shown to be dependant on the presence and concentration of certain cation species. Anion species appear to have little effect on streptodornase activity. In particular, the divalent cations magnesium and calcium are essential for optimum activity whilst sodium ions are inhibitory. The overall effect of ion concentration on streptodornase activity is a result of a complex interplay between the various ion species. These effects and conditions are described and a possible explanation for the observed interplay is offered.

Introduction: Osteoarthritis (OA) is a degenerative joint disease partially mediated by the catabolic cytokine TNF-α, leading to progressive and permanent degeneration of cartilage. Silymarin, an extract from the ripe seeds of Silybum marianum , possesses anti-inflammatory properties that could help resolve inflammation in chondrocytes. This study aims to assess the chondroprotection and anti-inflammatory effects of silymarin on TNF-α induced cell death, pro-inflammatory cytokine and matrix-metalloproteinase release. Methods: Human C-20/A4 chondrocytic cells were treated with silymarin (2–20 μM) alone or in the presence of dexamethasone (10 μM) for 30 min prior to TNF-α (60 pg/ml) stimulation for 6 h. Cell viability was determined by MTT assat. Cell free supernatants were collected to detect IL-6, IL-8 and MMP-1 release by ELISA. RT-PCR was used to detect IL-6, IL-8 and MMP-1 gene expression. Data are expressed as Mean ± SD of n = 4 determinations in triplicate. * P P

SUMMARYAlthough circulating anti-neutrophil cytoplasmic antibodies (ANCA) specific for myeloperoxidase (MPO) are strongly associated with the presence of vasculitis, they have been described in sera from patients with other conditions. High levels of anti-MPO antibodies can also persist in sera from patients with vasculitis despite the achievement of clinical remission. One possible interpretation is that a potentially pathogenic subset of anti-MPO antibodies exists, which is only present in patients with active vasculitis. We therefore compared the characteristics of anti-MPO antibodies in sera from patients with active vasculitis (n = 18) with those present in remission (n = 9) and in a disease control group (n = 10) without clinical evidence of vasculitis. The class, subclass and ability of anti-MPO antibodies from the three groups of patients to recognize three different conformational epitopes were analysed using ELISA-based techniques. The expression of an idiotope, designated 9G4, was also examined. Epitope recognition by anti-MPO antibodies from all patients tested was found to be similar. Sera from patients with active vasculitis showed an over-representation of IgG4 subclass anti-MPO antibodies and a more frequent presence of IgM class anti-MPO antibodies. In disease controls, IgG1 anti-MPO antibodies were predominant. In vitro, neutrophil activation by ANCA has been shown to be dependent on engagement of neutrophil FcγRIIa receptors following binding of these autoantibodies to surface-expressed ANCA antigens. We found that active vasculitis may be associated with the presence of circulating anti-MPO antibodies which do not significantly bind this receptor, suggesting that mechanisms other than those dependent on FcγRIIa binding should be explored. In addition, the expression of the 9G4 idiotope on anti-MPO antibodies in 60% (12/18) of patients with active vasculitis and 20% (2/10) of disease control patients may indicate a common origin for anti-MPO antibodies in different individuals.

An affinity chromatography method, using double stranded DNA, is described for the isolation of a deoxyribonuclease from the medicament Varidase, a wound cleansing preparation produced from the extracellular products of a species of Streptococcus. Using a complex buffer system, which includes EDTA, mercaptoethanol, bovine serum albumin and 50 mM NaCl, conditions are produced which allow binding of the nuclease to the chromatographic matrix without apparent degradation of the DNA. Increasing the NaCl concentration to 2 M results in the elution and isolation of a relatively non-specific endonuclease with a molecular mass of approximately 38 000 and a pI of 4.6. Experiments using ammonium sulphate for partial fractionation of the Varidase proteins, prior to chromatography, improved the yield but were not essential for obtaining a purified product. It is suggested that this technique should be readily applicable for the isolation of deoxyribonucleases from other sources.

RSNOs (S-nitrosothiols) regulate platelet and megakaryocyte function, and may act in vivo as a nitric oxide reservoir. There is a discrepancy between the spontaneous rate of NO release from different RSNO compounds and their pharmacological effects, implying that target cells may mediate biological activity either by metabolism of RSNOs or by displaying cell surface receptors. In the present study, we sought evidence for rapid cell-mediated metabolism of RSNOs. Exposure of platelets to GSNO (S-nitrosoglutathione) for as little as 5 s inhibited thrombin-induced platelet aggregation by >95%; however, AlbSNO (S-nitrosoalbumin) was much less effective over these short time periods. Incubation of 1 μM GSNO or AlbSNO with platelets and megakaryocytes resulted in a 25–34% loss of RSNO recoverable from the supernatant (P

This study investigated the role of urocortin (UCN), a member of the corticotrophin-releasing factor (CRF) family of peptides, in osteoclast maturation and function. We found that 10−7 M UCN significantly (P−9 M (P−7 M (P−7 M) but had no effect on osteoclast survival. Osteoclasts expressed mRNA encoding both UCN and the CRF receptor 2β subtype. Pre-osteoclasts however, expressed CRF receptor 2β alone. Unstimulated osteoclasts contained constitutively active cation channel currents with a unitary conductance of 3–4 pS, which were inhibited by over 70% with UCN (10−7 M). Compounds that regulate calcium signalling and energy status of the cell, both crucial for osteoclast activity were investigated. The non-selective cation channel blockers, lanthanum (La3+) and gadolinium (Gd3+), inhibited actin ring formation in osteoclasts, whereas modulators of voltage-dependent Ca2+channels and KATPchannels had no effect. These findings show for the first time that UCN is a novel anti-resorptive molecule that acts through a direct effect on osteoclasts and their precursor cells.

The activity of most deoxyribonuclease enzymes can be monitored by measuring the change in absorbance at 260 nm which accompanies the breakdown of the double-stranded structure of native DNA. An automated method for determining deoxyribonuclease activity, based on such an absorbance change, which can overcome problems of inhibition arising from the presence of inorganic cations, is described. Variations in inorganic cation concentration is a particular problem when measuring the activity of chromatographic fractions eluted via a salt gradient. A comparison is made between the automated and a manual method for the assay of deoxyribonuclease active constituents, of the medicament ‘Varidase’, eluted from a Cellex-D (Bio-Rad Laboratories Ltd) anionic exchange resin using a 0.05-1.0 M sodium chloride gradient.

The potential neuroprotective action of the corticotrophin-releasing factor-related peptide urocortin (UCN) was investigated in the rat 6-hydroxydopamine (6-OHDA) and lipopolysaccharide (LPS) paradigms of Parkinson's disease. UCN (20 fmol) was either given at the same time as (T = 0) or 7 days after (T = +7) intracerebral 6-OHDA or LPS injection. At 14 days after 6-OHDA or LPS injection, circling behaviour was measured following apomorphine challenge. Circling was significantly lower in rats given UCN at either T = 0 or T = +7 compared with animals given 6-OHDA or LPS and vehicle. Sham-treated rats showed no circling. Consistent with these observations, striatal dopamine concentrations were markedly higher in 6-OHDA/LPS + UCN rats vs. 6-OHDA/LPS + vehicle groups. Additionally, L-dihydroxyphenylalanine production by tyrosine hydroxylase was greatly reduced in the striata of 6-OHDA/LPS + vehicle rats, whereas this was not the case in rats coadministered UCN. Finally, the numbers of tyrosine hydroxylase-positive cells recorded in the substantia nigra of 6-OHDA/LPS + vehicle-treated animals were markedly lower than those of sham-operated or 6-OHDA/LPS + UCN rats. Critically, UCN was effective in reversing lesion-induced deficits when given either at the same time as or 7 days after the neurotoxic insult. To our knowledge, this is the first time that such an effect has been demonstrated in vivo. The apparent ability of UCN to arrest the progression of or even reverse nigral lesions once established suggests that pharmacological manipulation of this system could have substantial therapeutic utility.

The essential oil extracted from clove (Syzygium aromaticum) is used as a topical application to relieve pain and promote healing in herbal medicine and also finds use in the fragrance and flavouring industries. Clove oil has two major components, eugenol and β-caryophyllene, which constitute 78% and 13% of the oil, respectively. Clove oil and these components are generally recognized as ‘safe’, but the in-vitro study here demonstrates cytotoxic properties of both the oil and eugenol, towards human fibroblasts and endothelial cells. Clove oil was found to be highly cytotoxic at concentrations as low as 0.03% (v/v) with up to 73% of this effect attributable to eugenol. β-caryophyllene did not exhibit any cytotoxic activity, indicating that other cytotoxic components may also exist within the parent oil.

Lavender (Lavandula angustifolia) oil, chiefly composed of linalyl acetate (51%) and linalool (35%), is considered to be one of the mildest of known plant essential oils and has a history in wound healing. Concerns are building about the potential for irritant or allergenic skin reactions with the use of lavender oil. This study has demonstrated that lavender oil is cytotoxic to human skin cells in vitro (endothelial cells and fibroblasts) at a concentration of 0.25% (v/v) in all cell types tested (HMEC‐1, HNDF and 153BR). The major components of the oil, linalyl acetate and linalool, were also assayed under similar conditions for their cytotoxicity. The activity of linalool reflected that of the whole oil, indicating that linalool may be the active component of lavender oil. Linalyl acetate cytotoxicity was higher than that of the oil itself, suggesting suppression of its activity by an unknown factor in the oil. Membrane damage is proposed as the possible mechanism of action.

A 40 amino acid region of Oct-2 from amino acids 142 to 181 functions as an active repressor domain capable of inhibiting both basal activity and activation of promoters containing a TATA box, but not of those that contain an initiator element. Based on our observation that the equivalent region of the closely related Oct-1 factor does not act as an inhibitory domain, we have mutated specific residues in the Oct-2 domain in an attempt to probe their importance in repressor domain function. Although mutations of several residues have no or minimal effect, mutation of proline 175 to arginine abolishes the ability to inhibit both basal and activated transcription. In contrast, mutation of proline 174 to arginine confers upon the domain the ability to repress activation of an initiator-containing promoter by acidic activation domains, and also suppresses the effect of the proline 175 mutation. Hence, adjacent proline residues play key roles in the functioning of the inhibitory domain and in limiting its specificity to TATA-box-containing promoters.

Osteoarthritis (OA) is characterized by a loss of joint mobility and pain resulting from progressive destruction and loss of articular cartilage secondary to chondrocyte death and/ or senescence. Certain stimuli including nitric oxide (NO) and the pro-inflammatory cytokine tumor necrosis factor α (TNF-α have been implicated in this chondrocyte death and the subsequent accelerated damage to cartilage. In this study, we demonstrate that a corticotrophin releasing factor (CRF) family peptide, urocortin (Ucn), is produced by a human chondrocyte cell line, C-20/A4, and acts both as an endogenous survival signal and as a cytoprotective agent reducing the induction of apoptosis by NO but not TNF-α when added exogenously. Furthermore, treatment with the NO donor S-nitroso-N-acetyl-𝒟-ℒ-penicillamine upregulates chondrocyte Ucn expression, whereas treatment with TNF-α does not. The chondroprotective effects of Ucn are abolished by both specific ligand depletion (with an anti-Ucn antibody) and by CRF receptor blockade with the pan-CRFR antagonist α-helical CRH(9-41). CRFR expression was confirmed by reverse transcription-PCR with subsequent amplicon sequence analysis and demonstrates that C-20/A4 cells express both CRFR1 and CRFR2, specifically CRFR1α and CRFR2β. Protein expression of these receptors was confirmed by western blotting. The presence of both Ucn and its receptors in these cells, coupled with the induction of Ucn by NO, suggests the existence of an endogenous autocrine/paracrine chondroprotective mechanism against stimuli inducing chondrocyte apoptosis via the intrinsic/mitochondrial pathway.

Varidase used throughout the world for the topical treatment of purulent and suppurating wounds. The efficacy of the product is centred on two enzymes streptokinase and streptodornase; but these represent only a small proportion of the bulked solid. This article gives an overview of the preparation and mode of action of Varidase as well as showing some of the research and development that has gone into improving the assessment of its quality and composition.