Regulation of TNF-Induced Osteoclast Differentiation.

Increased osteoclast (OC) differentiation and activity is the critical event that results in bone loss and joint destruction in common pathological bone conditions, such as osteoporosis and rheumatoid arthritis (RA). RANKL and its decoy receptor, osteoprotegerin (OPG), control OC differentiation and activity. However, there is a specific concern of a rebound effect of denosumab discontinuation in treating osteoporosis. TNFα can induce OC differentiation that is independent of the RANKL/RANK system. In this review, we discuss the factors that negatively and positively regulate TNFα induction of OC formation, and the mechanisms involved to inform the design of new anti-resorptive agents for the treatment of bone conditions with enhanced OC formation. Similar to, and being independent of, RANKL, TNFα recruits TNF receptor-associated factors (TRAFs) to sequentially activate transcriptional factors NF-κB p50 and p52, followed by c-Fos, and then NFATc1 to induce OC differentiation. However, induction of OC formation by TNFα alone is very limited, since it also induces many inhibitory proteins, such as TRAF3, p100, IRF8, and RBP-j. TNFα induction of OC differentiation is, however, versatile, and Interleukin-1 or TGFß1 can enhance TNFα-induced OC formation through a mechanism which is independent of RANKL, TRAF6, and/or NF-κB. However, TNFα polarized macrophages also produce anabolic factors, including insulin such as 6 peptide and Jagged1, to slow down bone loss in the pathological conditions. Thus, the development of novel approaches targeting TNFα signaling should focus on its downstream molecules that do not affect its anabolic effect.

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 (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.

Poly(3-hydroxyoctanoate), a promising new material for cardiac tissue engineering.

Cardiac tissue engineering (CTE) is currently a prime focus of research because of an enormous clinical need. In the present 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. Results showed that the mechanical properties of the final patches were close to that of cardiac muscle. Biocompatibility of neat P(3HO) 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 into the patches. In addition, no deleterious effect was observed on adult cardiomyocyte 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 have 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. Copyright © 2016 John Wiley & Sons, Ltd.

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 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.

Melanocortin peptides protect chondrocytes from mechanically induced cartilage injury.

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 [DTRP(8)]-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 (1.14N, 6.47 kPa) delivered by a drop tower. Chondroprotective effects of α-MSH, [DTRP(8)]-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, [DTRP(8)]-γ-MSH and dexamethasone. Melanocortin pre-treatment of TNF-α-stimulated freshly isolated chondrocytes resulted in a bell-shaped inhibition in IL-1ß, IL-6 and IL-8, and elevation of IL-10 production. The MC3/4 antagonist, SHU9119, abrogated the effect of [DTRP(8)]-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.

Chondroprotective and anti-inflammatory role of melanocortin peptides in TNF-α activated human C-20/A4 chondrocytes.

BACKGROUND AND PURPOSE: Melanocortin MC(1) and MC(3 ) 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 MC(1) and MC(3) receptors on chondrocytes and the role of α-melanocyte-stimulating hormone (α-MSH) and the selective MC(3) receptor agonist, [DTRP(8) ]-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, [DTRP(8) ]-γ-MSH alone or in the presence of the MC(3/4) 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 MC(1,3) receptors. α-MSH and [DTRP(8) ]-γ-MSH treatment, for 30 min before TNF-α stimulation, provided a time-and-bell-shaped concentration-dependent decrease in pro-inflammatory cytokines (IL-1ß, 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 [DTRP(8) ]-γ-MSH treatment also inhibited TNF-α-induced caspase-3/7 activation and chondrocyte death. The effects of [DTRP(8) ]-γ-MSH, but not α-MSH, were abolished by the MC(3/4) receptor antagonist, SHU9119. CONCLUSION AND IMPLICATIONS: Activation of MC(1) /MC(3) 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 MC(1) /MC(3) receptors could be a viable approach for developing chondroprotective and anti-inflammatory therapies in rheumatoid and osteoarthritis.

Urocortin is a novel regulator of osteoclast differentiation and function through inhibition of a canonical transient receptor potential 1-like cation channel.

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<0.05) suppressed osteoclast differentiation from bone marrow precursor cells in culture and reduced the expression of several osteoclastic markers. Furthermore, UCN potently suppressed osteoclast bone resorption, by significantly inhibiting both the plan area of bone resorbed by osteoclasts and actin ring formation within osteoclasts at 10(-9) M (P<0.05), with complete inhibition at 10(-7) M (P<0.001). UCN also inhibited osteoclast motility (10(-7) M) but had no effect on osteoclast survival. Osteoclasts expressed mRNA encoding both UCN and the CRF receptor 2ß subtype. Pre-osteoclasts however, expressed CRF receptor 2ß 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 (La(3)(+)) and gadolinium (Gd(3)(+)), inhibited actin ring formation in osteoclasts, whereas modulators of voltage-dependent Ca(2)(+) channels and K(ATP) channels 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 corticotrophin-releasing factor-like peptide urocortin reverses key deficits in two rodent models of Parkinson's disease.

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.