Characterisation of pulpal responses to bacterial challenge and novel sntimicrobials for management of bacterial contamination of infected pulps.

Dental pain from pulpal infection and inflammation are the common cause of dental emergencies. Therefore, evaluation of modalities to treat them would be beneficial. This work aims to characterise and validate the previously developed ex vivo pulp infection model and make it reliable and reproducible in terms of quantifying cell viability, area of bacterial colonisation and expression of inflammatory markers. The method developed and used in this work demonstrated that SAG infection reduced cell viability and IL-10 levels. SAG infection also Increased area of SAG colonisation of pulp,expression of IL-β, TNF-a and IL-18 in a time depended manner compared to uninfected control pulp. These findings were consistent with clinical observations, making it a reliable model for the observed characteristics. There were differences in the response to two SAG strains, with similar overall trends, but with greater response to S. constellatus.Differences in response to the two strains was confirmed by evaluating the effect of the SAG supernatants using the model. The response observed confirmed the deleterious effects of the supernatant on cell viability, and increase in expression of inflammatory markers, with a greater response of pulp observed to S. constellatus. Analysing the supernatants of SAG strains revealed the difference could be due to hyaluronidase produced by S. constellatus.Triclosan’s anti-microbial and anti-inflammatory effect in the treatment of pulpal infection was demonstrated using the SAG model through increased cell viability, reduced area of colonisation and inflammatory marker expression levels form triclosan treatment. Triclosan showed no anti-hyaluronisase activity.A poly-microbial infection model was developed with E. faecalis and S. anginosus on tooth slices. It was observed that E. faecalis perpetuated S. anginosus growth in mixed culture and caused greater cell-death compared to S. anginosus mono-infection. The proportion of E.faecalis and its affinity to blood vessels could influence the response of the model

Enterococcus faecalis demonstrates pathogenicity through increased attachment in an ex vivo polymicrobial pulpal infection

This study investigated the host response to a polymicrobial pulpal infection consisting of Streptococcus anginosus and Enterococcus faecalis, bacteria commonly implicated in dental abscesses and endodontic failure, using a validated ex vivo rat tooth model. Tooth slices were inoculated with planktonic cultures of S. anginosus or E. faecalis alone or in co-culture at ratios of 50:50 and 90:10 S. anginosus to E. faecalis. Attachment was semi-quantified by measuring area covered by fluorescently labelled bacteria. Host response was established by viable histological cell counts and inflammatory response using RT-qPCR and immunohistochemistry. A significant reduction in cell viability was observed for single and polymicrobial infections, with no significant differences between infection types (≈2000cells/mm2 for infected pulps compared to ≈4000cells/mm2 for uninfected pulps). E. faecalis demonstrated significantly higher levels of attachment (6.5%) compared to S. anginosus alone (2.3%) and mixed species infections (3.4% for 50:50 and 2.3% for 90:10), with a remarkable affinity to the pulpal vasculature. Infections with E. faecalis demonstrated the greatest increase in TNF-α (47.1 fold for E. faecalis, 14.6 fold for S. anginosus, 60.1 fold for 50:50 and 25.0 fold for 90:10) and IL-1β expression (54.8 fold for E. faecalis, 8.8 fold for S. anginosus, 54.5 fold for 50:50 and 39.9 fold for 90:10) when compared to uninfected samples. Immunohistochemistry confirmed this with the majority of inflammation localised to the pulpal vasculature and odontoblast regions. Interestingly, E. faecalis supernatant and heat killed E. faecalis treatment was unable to induce the same inflammatory response, suggesting E. faecalis pathogenicity in pulpitis is linked to its greater ability to attach to the pulpal vasculature

Intra-Articular Injections of Polyphenols Protect Articular Cartilage from Inflammation-Induced Degradation: Suggesting a Potential Role in Cartilage Therapeutics

<div><p>Arthritic diseases, such as osteoarthritis and rheumatoid arthritis, inflict an enormous health care burden on society. Osteoarthritis, a degenerative joint disease with high prevalence among older people, and rheumatoid arthritis, an autoimmune inflammatory disease, both lead to irreversible structural and functional damage to articular cartilage. The aim of this study was to investigate the effect of polyphenols such as catechin, quercetin, epigallocatechin gallate, and tannic acid, on crosslinking type II collagen and the roles of these agents in managing <i>in vivo</i> articular cartilage degradation. The thermal, enzymatic, and physical stability of bovine articular cartilage explants following polyphenolic treatment were assessed for efficiency. Epigallocatechin gallate and tannic acid-treated explants showed >12 °C increase over native cartilage in thermal stability, thereby confirming cartilage crosslinking. Polyphenol-treated cartilage also showed a significant reduction in the percentage of collagen degradation and the release of glycosaminoglycans against collagenase digestion, indicating the increase physical integrity and resistance of polyphenol crosslinked cartilage to enzymatic digestion. To examine the <i>in vivo</i> cartilage protective effects, polyphenols were injected intra-articularly before (prophylactic) and after (therapeutic) the induction of collagen-induced arthritis in rats. The hind paw volume and histomorphological scoring was done for cartilage damage. The intra-articular injection of epigallocatechin gallate and tannic acid did not significantly influence the time of onset or the intensity of joint inflammation. However, histomorphological scoring of the articular cartilage showed a significant reduction in cartilage degradation in prophylactic- and therapeutic-groups, indicating that intra-articular injections of polyphenols bind to articular cartilage and making it resistant to degradation despite ongoing inflammation. These studies establish the value of intra-articular injections of polyphenol in stabilization of cartilage collagen against degradation and indicate the unique beneficial role of injectable polyphenols in protecting the cartilage in arthritic conditions.</p></div

Percentage of collagen degradation and release of GAGs from bovine AC (with and without polyphenols) treated with collagenase.

<p>^aData were analyzed using one-way ANOVA with Bonferroni post hoc test. The EGCG (P<0.01), TA (P<0.05), and CAT (P<0.05) treated groups showed significantly reduced percent degradation of collagen compared to controls. Similarly, the percent release of GAG in the EGCG- (P<0.01) and TA-treated (P<0.01) groups had significantly reduced (P-value: 0.0268) compared to controls but no statistical significance was observed in CAT and QUE. The values are represented as Mean ± SEM, n = 3.</p><p>(* indicates significant difference in comparison to control, P<0.05.)</p><p>Percentage of collagen degradation and release of GAGs from bovine AC (with and without polyphenols) treated with collagenase.</p

Compression analysis of AC.

<p>Compressive load at 50% compression of AC incubated 4 days without and with polyphenols 200-μM (CAT, QUE, EGCG, TA) prepared using PBS; figure shows day-4 results; no significant difference (P>0.05) was observed between the control and polyphenol-treated samples; values are represented in Mean ± SEM, n = 3.</p

Effect of prophylactic treatment of polyphenols on collagen-induced arthritis (CIA).

<p>(A) Hematoxylin and Eosin-stained sections and (B) Masson's Trichrome stained sections of tibiofemoral joints of rats; NC indicates Negative Control (i.e., normal joint without induction and treatment); remaining illustrations are from CIA joints: PPC—Prophylactic Positive Control; PE—Prophylactic EGCG; and PT—Prophylactic TA; (black arrow = disintegrated cartilage; green arrows = intact cartilage)</p

Differential scanning calorimetric (DSC) analysis.

<p>Thermograms of control (native) and polyphenol-treated (CAT, QUE, EGCG, and TA) bovine AC samples (representative picture)</p

Paw volume and cartilage degradation scoring of therapeutically treated groups.

<p>(A) Paw volume changes in therapeutically treated rat groups from day 1–43 (values analyzed using RM-ANOVA with Graph Pad prism; "***" indicates significant difference compared to negative control (NC) (P<0.001); "#" indicates significant difference compared to therapeutic positive control (TPC) (### represents P<0.001, ## represents P<0.01 and # represents P<0.05); values represented in Mean ± SEM, n = 12. (B) Cartilage degradation scores from histomorphological sections of therapeutically treated groups; "**" indicates significant difference compared to NC (P<0.01); statistically significant differences between TPC and TT or TE groups indicated as "#" (P<0.05); values represented in Mean ± SEM, n = 6.</p