Christopher Irwin

Design, characterisation and preliminary clinical evaluation of a novel mucoadhesive topical formulation containing tetracycline for the treatment of periodontal disease

This study describes the formulation, characterisation and preliminary clinical evaluation of mucoadhesive, semi-solid formulations containing hydroxyethylcellulose (HEC, 1-5%, w/w), polyvinylpyrrolidine (PVP, 2 or 3%, w/w), polycarbophil (PC, 1 or 3%, w/w) and tetracycline (5%, w/w, as the hydrochloride). Each formulation was characterised in terms of drug release, hardness, compressibility, adhesiveness (using a texture analyser in texture profile analysis mode), syringeability (using a texture analyser in compression mode) and adhesion to a mucin disc (measured as a detachment force using the texture analyser in tensile mode). The release exponent for the formulations ranged from 0.78+/-0.02 to 1. 27+/-0.07, indicating that drug release was non-diffusion controlled. Increasing the concentrations of each polymeric component significantly increased the time required for 10 and 30% release of the original mass of tetracycline, due to both increased viscosity and, additionally, the unique swelling properties of the formulations. Increasing concentrations of each polymeric component also increased the hardness, compressibility, adhesiveness, syringeability and mucoadhesion of the formulations. The effects on product hardness, compressibility and syringeability may be due to increased product viscosity and, hence, increased resistance to compression. Similarly, the effects of these polymers on adhesiveness/mucoadhesion highlight their mucoadhesive nature and, importantly, the effects of polymer state (particularly PC) on these properties. Thus, in formulations where the neutralisation of PC was maximally suppressed, adhesiveness and mucoadhesion were also maximal. Interestingly, statistical interactions were primarily observed between the effects of HEC and PC on drug release, mechanical and mucoadhesive properties. These were explained by the effects of HEC on the physical state of PC, namely swollen or unswollen. In the preliminary clinical evaluation, a formulation was selected that offered an appropriate balance of the above physical properties and contained 3% HEC, 3% PVP and 1% PC, in addition to tetracycline 5% (as the hydrochloride). The clinical efficacy of this (test) formulation was compared to an identical tetracycline-devoid (control) formulation in nine periodontal pockets (>/=5 mm depth). One week following administration of the test formulation, there was a significant improvement in periodontal health as identified by reduced numbers of sub-gingival microbial pathogens. Therefore, it can be concluded that, when used in combination with mechanical plaque removal, the tetracycline-containing semi-solid systems described in this study would augment such therapy by enhancing the removal of pathogens, thus improving periodontal health.

Human odontoblasts express functional thermo-sensitive TRP channels: implications for dentin sensitivity

&NA; Odontoblasts form the outermost cellular layer of the dental pulp where they have been proposed to act as sensory receptor cells. Despite this suggestion, evidence supporting their direct role in mediating thermo‐sensation and nociception is lacking. Transient receptor potential (TRP) ion channels directly mediate nociceptive functions, but their functional expression in human odontoblasts has yet to be elucidated. In the present study, we have examined the molecular and functional expression of thermo‐sensitive TRP channels in cultured odontoblast‐like cells and in native human odontoblasts obtained from healthy wisdom teeth. PCR and western blotting confirmed gene and protein expression of TRPV1, TRPA1 and TRPM8 channels. Immunohistochemistry revealed that these channels were localised to odontoblast‐like cells as determined by double staining with dentin sialoprotein (DSP) antibody. In functional assays, agonists of TRPV1, TRPA1 and TRPM8 channels elicited [Ca2+]i transients that could be blocked by relevant antagonists. Application of hot and cold stimuli to the cells also evoked rises in [Ca2+]i which could be blocked by TRP‐channel antagonists. Using a gene silencing approached we further confirmed a role for TRPA1 in mediating noxious cold responses in odontoblasts. We conclude that human odontoblasts express functional TRP channels that may play a crucial role in mediating thermal sensation in teeth. Cultured and native human odontoblasts express functional TRP channels that may play a crucial role in mediating thermal sensation in teeth.

Evaluation of the Ability of LL-37 to Neutralise LPS In Vitro and Ex Vivo

Background Human cathelicidin LL-37 is a cationic antimicrobial peptide (AMP) which possesses a variety of activities including the ability to neutralise endotoxin. In this study, we investigated the role of LPS neutralisation in mediating LL-37s ability to inhibit Pseudomonas aeruginosa LPS signalling in human monocytic cells. Methodology/Principal Findings Pre-treatment of monocytes with LL-37 significantly inhibited LPS-induced IL-8 production and the signalling pathway of associated transcription factors such as NF-κB. However, upon removal of LL-37 from the media prior to LPS stimulation, these inhibitory effects were abolished. These findings suggest that the ability of LL-37 to inhibit LPS signalling is largely dependent on extracellular LPS neutralisation. In addition, LL-37 potently inhibited cytokine production induced by LPS extracted from P. aeruginosa isolated from the lungs of cystic fibrosis (CF) patients. In the CF lung, polyanionic molecules such as glycosaminoglycans (GAGs) and DNA bind LL-37 and impact negatively on its antibacterial activity. In order to determine whether such interactions interfere with the LPS neutralising ability of LL-37, the status of LL-37 and its ability to bind LPS in CF sputum were investigated. Overall our findings suggest that in the CF lung, the ability of LL-37 to bind LPS and inhibit LPS-induced IL-8 production is attenuated as a result of binding to DNA and GAGs. However, LL-37 levels and its concomitant LPS-binding activity can be increased with a combination of DNase and GAG lyase (heparinase II) treatment. Conclusions/Significance Overall, these findings suggest that a deficiency in available LL-37 in the CF lung may contribute to greater LPS-induced inflammation during CF lung disease.

Phenotypic differences between oral and skin fibroblasts in wound contraction and growth factor expression.

Wounds of the oral mucosa heal in an accelerated fashion with reduced scarring compared with cutaneous wounds. The differences in healing outcome between oral mucosa and skin could be because of phenotypic differences between the respective fibroblast populations. This study compared paired mucosal and dermal fibroblasts in terms of collagen gel contraction, α‐smooth muscle actin expression (α‐SMA), and production of the epithelial growth factors: keratinocyte growth factor (KGF) and hepatocyte growth factor/scatter factor (HGF). The effects of transforming growth factor ‐β1 and ‐β3 on each parameter were also determined. Gel contraction in floating collagen lattices was determined over a 7‐day period. α‐SMA expression by fibroblasts was determined by Western blotting. KGF and HGF expression were determined by an enzyme‐linked immunosorbent assay. Oral fibroblasts induced accelerated collagen gel contraction, yet surprisingly expressed lower levels of α‐SMA. Oral cells also produced significantly greater levels of both KGF and HGF than their dermal counterparts. Transforming growth factor‐β1 and ‐β3, over the concentration range of 0.1–10 ng/mL, had similar effects on cell function, stimulating both gel contraction and α‐SMA production, but inhibiting KGF and HGF production by both cell types. These data indicate phenotypic differences between oral and dermal fibroblasts that may well contribute to the differences in healing outcome between these two tissues.

Neuropeptides regulate expression of angiogenic growth factors in human dental pulp fibroblasts.

INTRODUCTION Neuropeptides play an important role in inflammation and repair and have been implicated in mediating angiogenesis. Pulp fibroblasts express neuropeptide receptors, and the aim of this research was to investigate whether neuropeptides could regulate angiogenic growth factor expression in vitro METHODS An angiogenic array was used to determine the levels of 10 angiogenic growth factors expressed by human pulp fibroblasts. RESULTS Pulp fibroblasts were shown to express angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin-binding epidermal growth factor, hepatocyte growth factor, leptin, platelet-derived growth factor, placental growth factor, and vascular endothelial growth factor. Furthermore, the neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide Y altered angiogenic growth factor expression in vitro. CONCLUSIONS The regulation of angiogenic growth factor expression by neuropeptides suggests a novel role for neuropeptides in pulpal inflammation and repair.

Strawberry gums: a clinicopathological manifestation diagnostic of Wegener's granulomatosis?

AIMS--To highlight an uncommon but characteristic gingival lesion associated with Wegeners granulomatosis, emphasising the presence of pseudoepitheliomatous hyperplasia, microabscesses, and multinucleate giant cells; and the paucity of the currently accepted histopathological criteria of Wegeners granulomatosis--namely necrosis, vasculitis, and granulomata. METHODS--The histopathological features of a gingival biopsy specimen from a case of Wegeners granulomatosis limited to the upper aerodigestive tract in a 36 year old woman were compared with those of 14 similar reported cases. RESULTS--Pseudoepitheliomatous hyperplasia, microabscesses, and multinucleate giant cells were recorded as present in almost all cases of gingival Wegeners granulomatosis. Necrosis, vasculitis, and granuloma formation were present in only a few cases. CONCLUSIONS--The combination of pseudoepitheliomatous hyperplasia, microabscesses, and multinucleate giant cells in a gingival biopsy specimen from erythematous, swollen gums, clinically resembling over-ripe strawberries, in a patient with a severe systemic upset, is so typical of Wegeners granulomatosis as to be virtually diagnostic. Recognition of this characteristic lesion may aid early diagnosis and treatment in cases where other diagnostic variables are absent.

Human Dental Pulp Fibroblasts Express the “Cold-sensing” Transient Receptor Potential Channels TRPA1 and TRPM8

INTRODUCTION Transient receptor potential (TRP) channels comprise a group of nonselective calcium-permeable cationic channels, which are polymodal sensors of environmental stimuli such as thermal changes and chemicals. TRPM8 and TRPA1 are cold-sensing TRP channels activated by moderate cooling and noxious cold temperatures, respectively. Both receptors have been identified in trigeminal ganglion neurones, and their expression in nonneuronal cells is now the focus of much interest. The aim of this study was to investigate the molecular and functional expression of TRPA1 and TRPM8 in dental pulp fibroblasts. METHODS Human dental pulp fibroblasts were derived from healthy molar teeth. Gene and protein expression was determined by polymerase chain reaction and Western blotting. Cellular localization was investigated by immunohistochemistry, and TRP functionality was determined by Ca(2+) microfluorimetry. RESULTS Polymerase chain reaction and Western blotting showed gene and protein expression of both TRPA1 and TRPM8 in fibroblast cells in culture. Immunohistochemistry studies showed that TRPA1 and TRPM8 immunoreactivity co-localized with the human fibroblast surface protein. In Ca(2+) microfluorimetry studies designed to determine the functionality of TRPA1 and TRPM8 in pulp fibroblasts, we showed increased intracellular calcium ([Ca(2+)](i)) in response to the TRPM8 agonist menthol, the TRPA1 agonist cinnamaldehyde, and to cool and noxious cold stimuli, respectively. The responses to agonists and thermal stimuli were blocked in the presence of specific TRPA1 and TRPM8 antagonists. CONCLUSIONS Human dental pulp fibroblasts express TRPA1 and TRPM8 at the molecular, protein, and functional levels, indicating a possible role for fibroblasts in mediating cold responses in human teeth.

Matrix Metalloproteinase-3 Differences in Oral and Skin Fibroblasts

While skin wounds heal by scarring, wounds of oral mucosa show privileged healing with minimal scar formation. Our hypothesis was that phenotypic differences between oral and skin fibroblasts underlie these differences in healing. The aims of this study were to compare MMP-3 expression by oral and skin fibroblasts and investigate a role for MMP-3 in mediating collagen gel contraction. Oral fibroblasts induced significantly greater gel contraction than did paired skin cells. Inhibition of MMP activity significantly inhibited gel contraction by both cell types. Specific inhibition of MMP-3 activity reduced gel contraction by oral, but not skin, fibroblasts. Oral fibroblasts produced significantly higher levels of MMP-3 than did skin fibroblasts at all levels studied. TGF-β1 and -β3 isoforms stimulated MMP-3 expression at mRNA, protein, and activity levels by both fibroblast populations. Results suggest that increased MMP-3 production by oral fibroblasts may underlie the differences in wound-healing outcome seen in skin and oral mucosa.

Dental manifestations of pregnancy

• Pregnancy has significant effects on the periodontal tissues and pregnancy gingivitis is a common manifestation of this. • The host response and oral flora are affected and tooth surface loss and mobility may develop. • Further research is required to establish the association between periodontal health and adverse pregnancy outcome.

LL‐37 in periodontal health and disease and its susceptibility to degradation by proteinases present in gingival crevicular fluid

AIM To determine the levels of LL-37 in and its susceptibility to degradation by components of gingival crevicular fluid (GCF) in periodontal health and disease. MATERIALS AND METHODS Levels of LL-37 in GCF from periodontitis patients and periodontally healthy subjects were determined by ELISA. In addition, degradation of synthetic/exogenous LL-37 by components of GCF in the presence and absence of inhibitors was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. RESULTS The concentration of native LL-37 in GCF from Porphyromonas gingivalis positive (Pg+) and P. gingivalis negative (Pg-) sites in periodontitis patients was significantly higher than in GCF from healthy subjects. When synthetic LL-37 was added to healthy GCF, the peptide was not degraded. Conversely, GCF from Pg+ sites rapidly degraded synthetic LL-37 which was prevented in the presence of Arg- and Lys- gingipain inhibitors. Synthetic LL-37 was degraded more slowly by GCF from Pg- sites. CONCLUSIONS LL-37 is detectable in GCF in periodontal health and disease. The rapid degradation of synthetic LL-37 in periodontitis GCF, particularly in Pg+ sites, limits its role as a potential therapeutic in the gingival crevice. These results highlight the need to design stable peptide mimetics of LL-37 as future therapeutics in periodontitis.