Epoxytiglianes induce keratinocyte wound healing responses via classical protein kinase C activation to promote skin re-epithelialization.

Epoxytiglianes are a novel class of diterpene esters. The prototype epoxytigliane, EBC-46 (tigilanol tiglate), is a potent anti-cancer agent in clinical development for local treatment of a range of human and animal tumors. EBC-46 also consistently promotes wound re-epithelialization at the treatment sites, mediated via activation of classical protein kinase C (PKC) isoforms. We have previously shown that epoxytiglianes stimulate proliferative and wound repopulation responses in immortalized human skin keratinocytes (HaCaTs) in vitro, abrogated by pan-PKC inhibitor, bisindolylmaleimide-1. In this study, we further investigate the specific PKC isoforms responsible for inducing such wound healing responses, following HaCaT treatment with 1.51 nM-15.1 µM EBC-46 or analogue, EBC-211. Classical PKC inhibition by GÓ¦6976 (1 µM), significantly attenuated epoxytigliane induced, HaCaT proliferation and wound repopulation at all epoxytigliane concentrations. PKC-ßI/-ßII isoform inhibition by enzastaurin (1 µM), significantly inhibited HaCaT proliferation and wound repopulation responses induced by both epoxytiglianes, especially at 1.51-151 nM. PKC-α inhibitor, Ro 31-8220 mesylate (10 nM), exerted lesser inhibitory effects on HaCaT responses. Epoxytigliane changes in key keratin (KRT17) and cell cycle (cyclin B1, CDKN1A) protein levels were partly attenuated by GÓ¦6976 and enzastaurin. GÓ¦6976 also inhibited increases in matrix metalloproteinase (MMP-1, MMP-7, MMP-10) activities. Phospho-PKC (p-PKC) studies confirmed that epoxytiglianes transiently activated classical PKC isoforms (p-PKCα, p-PKC-ßI/-ßII, p-PKCγ) in a dose- and time-dependent manner. By identifying how epoxytiglianes stimulate classical PKCs to facilitate keratinocyte healing responses and re-epithelialization, these findings support further epoxytigliane development as topical therapeutics for clinical situations involving impaired re-epithelialization, such as non-healing wounds in skin.

Tigilanol Tiglate-Induced Changes in Secretome Profiles Alter C-Met Phosphorylation and Cell Surface Protein Expression in H357 Head and Neck Cancer Cells.

Tigilanol tiglate (TT, also known as EBC-46) is a novel, plant-derived diterpene ester possessing anticancer and wound-healing properties. Here, we show that TT-evoked PKC-dependent S985 phosphorylation of the tyrosine kinase MET leads to subsequent degradation of tyrosine phosphorylated p-Y1003 and p-Y1234/5 MET species. PKC inhibition with BIM-1 blocked S985 phosphorylation of MET and led to MET cell surface accumulation. Treatment with metalloproteinase inhibitors prevented MET-ECD release into cell culture media, which was also blocked by PKC inhibitors. Furthermore, unbiased secretome analysis, performed using TMT-technology, identified additional targets of TT-dependent release of cell surface proteins from H357 head and neck cancer cells. We confirm that the MET co-signalling receptor syndecan-1 was cleaved from the cell surface in response to TT treatment. This was accompanied by rapid cleavage of the cellular junction adhesion protein Nectin-1 and the nerve growth factor receptor NGFRp75/TNFR16. These findings, that TT is a novel negative regulator of protumorigenic c-MET and NGFRp75/TNFR16 signalling, as well as regulating Nectin-1-mediated cell adhesion, further contribute to our understanding of the mode of action and efficacy of TT in the treatment of solid tumours.

MicroRNA-155 (miR-155) as an accurate biomarker of periodontal status and coronary heart disease severity: a case-control study.

BACKGROUND: Increasing evidence supports associations between periodontal disease and coronary heart disease (CHD). This case-control study evaluated whether inflammatory regulator, microRNA-155 (miR-155), could be utilised as a biomarker of periodontitis and/or CHD. METHODS: Of 120 participants, 30 patients had clinically healthy periodontium (controls, C), 30 patients had generalized periodontitis (P), 30 patients had CHD and clinically healthy periodontium (AS-C); and 30 patients had CHD with generalized periodontitis (AS-P). Patient demographic and periodontal characteristics (plaque index, bleeding on probing, probing pocket depth and clinical attachment loss), were collected. Patient whole blood and saliva levels of miR-155 and pro-inflammatory cytokine (interleukin-1ß), were quantified by quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). One-way ANOVA with post-hoc Tukey test was used to determine differences among the four groups. Chi Square test was used for participant gender comparisons. Pearson correlation tests and multiple linear regression analyses were used to assess associations between the demographic and clinical variables analysed, versus IL-1ß and miR-155 levels. miR-155 and IL-1ß accuracy in differentiating healthy versus other patient groups were analysed using receiver operating characteristic (ROC) curves, by calculating area under the curve (AUC) values and sensitivity and specificity cut-off points using Youden's index. Statistical tests of sensitivity and specificity were conducted using the McNemar test. RESULTS: Whole blood miR-155 levels were elevated in periodontitis/non-periodontitis patients with CHD (AS-P, AS-C), and periodontitis patients alone (P) (p < 0.001). Receiver operating characteristic (ROC) and area under the curve (AUC) analyses confirmed miR-155 accuracy in discriminating P, AS-C and AS-P groups (AUC 0.6861-0.9944, p < 0.0001-0.05), coupled with high sensitivity (76.7-100.0%), specificity (53.3-96.7%) and cut-off points (> 0.955- > 2.915 a.u.; p < 0.0001). miR-155 levels further distinguished between CHD (AS-C, AS-P) and periodontitis (P) patients (AUC ≥ 0.8378, sensitivity ≥ 88.7%, specificity ≥ 73.3%, cut-off > 2.82 a.u; p < 0.0001), and between AS-C and AS-P patients (AUC 0.7578, sensitivity 80.0%, specificity 50.0%, cut-off > 7.065 a.u; p < 0.001). Subsequent analyses identified positive correlations between miR-155 and the various patient demographics, salivary interleukin-1ß and periodontal parameters assessed. CONCLUSIONS: This study advocates miR-155 as an accurate diagnostic/prognostic biomarker of periodontitis and/or CHD severity, thereby improving detection and treatment for both conditions.

Role of Enzymic Antioxidants in Mediating Oxidative Stress and Contrasting Wound Healing Capabilities in Oral Mucosal/Skin Fibroblasts and Tissues.

Unlike skin, oral mucosal wounds are characterized by rapid healing and minimal scarring, attributable to the "enhanced" healing properties of oral mucosal fibroblasts (OMFs). As oxidative stress is increasingly implicated in regulating wound healing outcomes, this study compared oxidative stress biomarker and enzymic antioxidant profiles between patient-matched oral mucosal/skin tissues and OMFs/skin fibroblasts (SFs) to determine whether superior oral mucosal antioxidant capabilities and reduced oxidative stress contributed to these preferential healing properties. Oral mucosa and skin exhibited similar patterns of oxidative protein damage and lipid peroxidation, localized within the lamina propria/dermis and oral/skin epithelia, respectively. SOD1, SOD2, SOD3 and catalase were primarily localized within epithelial tissues overall. However, SOD3 was also widespread within the lamina propria localized to OMFs, vasculature and the extracellular matrix. OMFs were further identified as being more resistant to reactive oxygen species (ROS) generation and oxidative DNA/protein damage than SFs. Despite histological evaluation suggesting that oral mucosa possessed higher SOD3 expression, this was not fully substantiated for all OMFs examined due to inter-patient donor variability. Such findings suggest that enzymic antioxidants have limited roles in mediating privileged wound healing responses in OMFs, implying that other non-enzymic antioxidants could be involved in protecting OMFs from oxidative stress overall.

Evidence for Natural Products as Alternative Wound-Healing Therapies.

Chronic, non-healing wounds represent a significant area of unmet medical need and are a growing problem for healthcare systems around the world. They affect the quality of life for patients and are an economic burden, being difficult and time consuming to treat. They are an escalating problem across the developed world due to the increasing incidence of diabetes and the higher prevalence of ageing populations. Effective treatment options are currently lacking, and in some cases chronic wounds can persist for years. Some traditional medicines are believed to contain bioactive small molecules that induce the healing of chronic wounds by reducing excessive inflammation, thereby allowing re-epithelisation to occur. Furthermore, many small molecules found in plants are known to have antibacterial properties and, although they lack the therapeutic selectivity of antibiotics, they are certainly capable of acting as topical antiseptics when applied to infected wounds. As these molecules act through mechanisms of action distinct from those of clinically used antibiotics, they are often active against antibiotic resistant bacteria. Although there are numerous studies highlighting the effects of naturally occurring small molecules in wound-healing assays in vitro, only evidence from well conducted clinical trials can allow these molecules or the remedies that contain them to progress to the clinic. With this in mind, we review wound-healing natural remedies that have entered clinical trials over a twenty-year period to the present. We examine the bioactive small molecules likely to be in involved and, where possible, their mechanisms of action.

Synergistic activity of pomegranate rind extract and Zn (II) against Candida albicans under planktonic and biofilm conditions, and a mechanistic insight based upon intracellular ROS induction.

Candida albicans (C. albicans) is an opportunistic pathogen, which causes superficial infection and can lead to mortal systemic infections, especially in immunocompromised patients. The incidence of C. albicans infections is increasing and there are a limited number of antifungal drugs used in treatment. Therefore, there is an urgent need for new and alternative antifungal drugs. Pomegranate rind extract (PRE) is known for its broad-spectrum antimicrobial activities, including against C. albicans and recently, PRE and Zn (II) have been shown to induce synergistic antimicrobial activity against various microbes. In this study, the inhibitory activities of PRE, Zn (II) and PRE in combination with Zn (II) were evaluated against C. albicans. Antifungal activities of PRE and Zn (II) were evaluated using conventional microdilution methods and the interaction between these compounds was assessed by in vitro checkerboard and time kill assays in planktonic cultures. The anti-biofilm activities of PRE, Zn (II) and PRE in combination with Zn (II) were assessed using confocal laser scanning microscopy, with quantitative analysis of biofilm biomass and mean thickness analysed using COMSTAT2 analysis. In addition, antimicrobial interactions between PRE and Zn (II) were assayed in terms reactive oxygen species (ROS) production by C. albicans. PRE and Zn (II) showed a potent antifungal activity against C. albicans, with MIC values of 4 mg/mL and 1.8 mg/mL, respectively. PRE and Zn (II) in combination exerted a synergistic antifungal effect, as confirmed by the checkerboard and time kill assays. PRE, Zn (II) and PRE and Zn (II) in combination gave rise to significant reductions in biofilm biomass, although only PRE caused a significant reduction in mean biofilm thickness. The PRE and Zn (II) in combination caused the highest levels of ROS production by C. albicans, in both planktonic and biofilm forms. The induction of excess ROS accumulation in C. albicans may help explain the synergistic activity of PRE and Zn (II) in combination against C. albicans in both planktonic and biofilm forms. Moreover, the data support the potential of the PRE and Zn (II) combination as a novel potential anti-Candida therapeutic system.

Anti-Fibrotic Potential of Tomentosenol A, a Constituent of Cerumen from the Australian Native Stingless Bee, Tetragonula carbonaria.

Bioactivity-guided fractionation was used to isolate two compounds, tomentosenol A (1) and torellianone A (2), from a cerumen extract from Tetragonula carbonaria. The anti-fibrotic activity of these compounds was examined using human cultured neonatal foreskin fibroblasts (NFF) and immortalised keratinocytes (HaCaTs). Tomentosenol A (1), inhibited NFF and HaCaT cell proliferation and prevented NFF and HaCaT scratch wound repopulation at 12.5-25 µM concentrations. These inhibitory effects were associated with reduced cell viability, determined by tetrazolium dye (MTT) and sulforhodamine B (SRB) assays. Compound 1 further inhibited transforming growth factor-ß1 (TGF-ß1)-stimulated, NFF-myofibroblast differentiation and soluble collagen production; and was an effective scavenger of the model oxidant, 2,2-diphenyl-1-picrylhydrazyl (DPPH·), with an EC50 value of 44.7 ± 3.1 µM. These findings reveal significant anti-fibrotic potential for cerumen-derived tomentosenol A (1).

Ficus septica exudate, a traditional medicine used in Papua New Guinea for treating infected cutaneous ulcers: in vitro evaluation and clinical efficacy assessment by cluster randomised trial.

BACKGROUND AND OBJECTIVES: Infected cutaneous ulcers are major health problems for children living in rural areas of Papua New Guinea. The inaccessibility of affected populations and lack of access to basic healthcare, make a local plant-based therapy an attractive treatment option. We assessed Ficus septica exudate in biological assays relevant to wound healing. We then carried out a clinical trial to determine the exudate's efficacy in healing small cutaneous ulcers compared with Savlon antiseptic cream, and soap and water washing. METHODS: Pre-clinical in vitro assessment of the exudate was carried out using assays to monitor the pro-inflammatory responses of M1 macrophages and neutrophils, antibacterial assays using known ulcer pathogens, an Ames test for mutagenicity and LC-MS chemical analysis of the exudate. An open label cluster-randomised clinical trial was performed, enrolling participants from three different clusters with skin lesions less than 1 cm in diameter. Each cluster comprising 50 participants was randomly assigned to one of three treatment arms namely topical exudate, topical Savlon antiseptic cream, and standard care (soap and water treatment), all administered daily for 2 days. The primary outcome was clinical healing/improvement measured at days 7 and 14, assessed by three dermatologists using blinded photographs. The primary analysis was assessed as non-inferiority of F. septica treatment based on the risk difference for healing/improvement. RESULTS: In vitro, the exudate which is rich in the alkaloid ficuseptine, was found to be non-mutagenic whilst also inhibiting pro-inflammatory responses and exhibiting antibacterial activity. When administered to participants enrolled in the clinical trial, no significant differences were observed between the healing efficacy of F. septica exudate and the two comparator treatments (Savlon antiseptic cream and soap/water treatment). At day 14, but not at day 7, the efficacy of F. septica exudate for healing/improving the ulcers was non-inferior to Savlon antiseptic cream or water/soap treatment. CONCLUSIONS: F. septica exudate is non-mutagenic and has both bactericidal and anti-inflammatory properties. When applied topically to small cutaneous ulcers, the exudate has a healing effect that is non-inferior to Savlon antiseptic cream and standard treatment with soap and water at day 14. Our findings, which should be confirmed in larger clinical trials, have important public health implications.

Dental Pulp Stem Cell Heterogeneity: Finding Superior Quality "Needles" in a Dental Pulpal "Haystack" for Regenerative Medicine-Based Applications.

Human dental pulp stem/stromal cells (hDPSCs) derived from the permanent secondary dentition are recognised to possess certain advantageous traits, which support their potential use as a viable source of mesenchymal stem/stromal cells (MSCs) for regenerative medicine-based applications. However, the well-established heterogeneous nature of hDPSC subpopulations, coupled with their limited numbers within dental pulp tissues, has impeded our understanding of hDPSC biology and the translation of sufficient quantities of these cells from laboratory research, through successful therapy development and clinical applications. This article reviews our current understanding of hDPSC biology and the evidence underpinning the molecular basis of their heterogeneity, which may be exploited to distinguish individual subpopulations with specific or superior characteristics for regenerative medicine applications. Pertinent unanswered questions which still remain, regarding the developmental origins, hierarchical organisation, and stem cell niche locations of hDPSC subpopulations and their roles in hDPSC heterogeneity and functions, will further be explored. Ultimately, a greater understanding of how key features, such as specific cell surface, senescence and other relevant genes, and protein and metabolic markers, delineate between hDPSC subpopulations with contrasting stemness, proliferative, multipotency, immunomodulatory, anti-inflammatory, and other relevant properties is required. Such knowledge advancements will undoubtedly lead to the development of novel screening, isolation, and purification strategies, permitting the routine and effective identification, enrichment, and expansion of more desirable hDPSC subpopulations for regenerative medicine-based applications. Furthermore, such innovative measures could lead to improved cell expansion, manufacture, and banking procedures, thereby supporting the translational development of hDPSC-based therapies in the future.

A Time-Kill Assay Study on the Synergistic Bactericidal Activity of Pomegranate Rind Extract and Zn (II) against Methicillin-Resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa.

There is a need for new antimicrobial systems due to increased global resistance to current antimicrobials. Pomegranate rind extract (PRE) and Zn (II) ions both possess a level of antimicrobial activity and work has previously shown that PRE/Zn (II) in combination possesses synergistic activity against Herpes simplex virus and Micrococcus luteus. Here, we determined whether such synergistic activity extended to other, more pathogenic, bacteria. Reference strains of methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa were cultured and subjected to challenge by PRE, Zn (II), or PRE + Zn (II), in time-kill assays. Data were obtained independently by two researchers using different PRE preparations. Statistically significant synergistic activity for PRE + Zn (II) was shown for all four bacterial strains tested compared to untreated controls, although the extent of efficacy and timescales varied. Zn (II) exerted activity and at 1 h, it was not possible to distinguish with PRE + Zn (II) combination treatment in all cases. PRE alone showed low activity against all four bacteria. Reproducible synergistic bactericidal activity involving PRE and Zn (II) has been confirmed. Potential mechanisms are discussed. The development of a therapeutic system that possesses demonstrable antimicrobial activity is supported which lends itself particularly to topical delivery applications, for example MRSA infections.

Modification of gingival proteoglycans by reactive oxygen species: potential mechanism of proteoglycan degradation during periodontal diseases.

Reactive oxygen species (ROS) overproduction and oxidative stress are increasingly being implicated in the extracellular matrix (ECM) degradation associated with chronic inflammatory conditions, such as periodontal diseases. The present study investigated the effects of ROS exposure on the proteoglycans of gingival tissues, utilizing an in vitro model system comprised of supra-physiological oxidant concentrations, to ascertain whether gingival proteoglycan modification and degradation by ROS contributed to the underlying mechanisms of ECM destruction during active gingivitis. Proteoglycans were purified from ovine gingival tissues and exposed to increasing H2O2 concentrations or a hydroxyl radical (·OH) flux for 1 h or 24 h, and ROS effects on proteoglycan core proteins and sulfated glycosaminoglycan (GAG) chains were assessed. ROS were capable of degrading gingival proteoglycans, with ·OH species inducing greater degradative effects than H2O2 alone. Degradative effects were particularly manifested as amino acid modification, core protein cleavage, and GAG chain depolymerization. Proteoglycan core proteins were more susceptible to degradation than GAG chains with H2O2 alone, although core proteins and GAG chains were both extensively degraded by ·OH species. Proteoglycan exposure to ·OH species for 24 h induced significant core protein amino acid modification, with decreases in glutamate, proline, isoleucine, and leucine; and concomitant increases in serine, glycine, and alanine residues. As clinical reports have previously highlighted proteoglycan core protein degradation during chronic gingivitis, whereas their sulfated GAG chains remain relatively intact, these findings potentially provide further evidence to implicate ROS in the pathogenesis of active gingivitis, complementing the enzymic mechanisms of periodontal tissue destruction already established.

Myofibroblasts: Function, Formation, and Scope of Molecular Therapies for Skin Fibrosis.

Myofibroblasts are contractile, α-smooth muscle actin-positive cells with multiple roles in pathophysiological processes. Myofibroblasts mediate wound contractions, but their persistent presence in tissues is central to driving fibrosis, making them attractive cell targets for the development of therapeutic treatments. However, due to shared cellular markers with several other phenotypes, the specific targeting of myofibroblasts has long presented a scientific and clinical challenge. In recent years, myofibroblasts have drawn much attention among scientific research communities from multiple disciplines and specialisations. As further research uncovers the characterisations of myofibroblast formation, function, and regulation, the realisation of novel interventional routes for myofibroblasts within pathologies has emerged. The research community is approaching the means to finally target these cells, to prevent fibrosis, accelerate scarless wound healing, and attenuate associated disease-processes in clinical settings. This comprehensive review article describes the myofibroblast cell phenotype, their origins, and their diverse physiological and pathological functionality. Special attention has been given to mechanisms and molecular pathways governing myofibroblast differentiation, and updates in molecular interventions.

Synergistic In Vitro Antimicrobial Activity of Pomegranate Rind Extract and Zinc (II) against Micrococcus luteus under Planktonic and Biofilm Conditions.

Infectious diseases caused by microbial biofilms are a major clinical problem, and new antimicrobial agents that can inhibit biofilm formation and eradicate pre-formed biofilms are urgently needed. Pomegranate extracts are a well-established folkloric medicine and have been used in the treatment of infectious diseases since ancient times, whilst the addition of metal ions, including zinc (II), has enhanced the antimicrobial activity of pomegranate. Micrococcus luteus is generally a non-pathogenic skin commensal bacterium, although it can act as an opportunistic pathogen and cause serious infections, particularly involving catheterization and comorbidities. The aims of this study were to evaluate the holistic activity of pomegranate rind extract (PRE), Zn (II), and PRE/Zn (II) individually and in combination against M. luteus under both planktonic and biofilm conditions. Antimicrobial activity was detected in vitro using the broth dilution method, and synergistic activity was determined using checkerboard and time-kill assays. Effects on biofilm formation and eradication were determined by crystal violet and BacLightTM Live/Dead staining. PRE and Zn (II) exerted antimicrobial activity against M. luteus under both planktonic and biofilm conditions. After 4 h, potent synergistic bactericidal activity was also found when PRE and Zn (II) were co-administered under planktonic conditions (log reductions: PRE 1.83 ± 0.24, Zn (II) 3.4 ± 0.08, and PRE/Zn (II) 6.88 ± 1.02; p < 0.0001). In addition, greater heterogeneity was induced in the structure of M. luteus biofilm using the PRE/Zn (II) combination compared to when PRE and Zn (II) were applied individually. The activity of PRE and the PRE/Zn (II) combination could offer a novel antimicrobial therapy for the treatment of disease-associated infections caused by M. luteus and potentially other bacteria.

Differential SOD2 and GSTZ1 profiles contribute to contrasting dental pulp stem cell susceptibilities to oxidative damage and premature senescence.

BACKGROUND: Dental pulp stem cells (DPSCs) are increasingly being advocated as viable cell sources for regenerative medicine-based therapies. However, significant heterogeneity in DPSC expansion and multi-potency capabilities are well-established, attributed to contrasting telomere profiles and susceptibilities to replicative senescence. As DPSCs possess negligible human telomerase (hTERT) expression, we examined whether intrinsic differences in the susceptibilities of DPSC sub-populations to oxidative stress-induced biomolecular damage and premature senescence further contributed to this heterogeneity, via differential enzymic antioxidant capabilities between DPSCs. METHODS: DPSCs were isolated from human third molars by differential fibronectin adhesion, and positive mesenchymal (CD73/CD90/CD105) and negative hematopoietic (CD45) stem cell marker expression confirmed. Isolated sub-populations were expanded in H2O2 (0-200 µM) and established as high or low proliferative DPSCs, based on population doublings (PDs) and senescence (telomere lengths, SA-ß-galactosidase, p53/p16INK4a/p21waf1/hTERT) marker detection. The impact of DPSC expansion on mesenchymal, embryonic, and neural crest marker expression was assessed, as were the susceptibilities of high and low proliferative DPSCs to oxidative DNA and protein damage by immunocytochemistry. Expression profiles for superoxide dismutases (SODs), catalase, and glutathione-related antioxidants were further compared between DPSC sub-populations by qRT-PCR, Western blotting and activity assays. RESULTS: High proliferative DPSCs underwent > 80PDs in culture and resisted H2O2-induced senescence (50-76PDs). In contrast, low proliferative sub-populations exhibited accelerated senescence (4-32PDs), even in untreated controls (11-34PDs). While telomere lengths were largely unaffected, certain stem cell marker expression declined with H2O2 treatment and expansion. Elevated senescence susceptibilities in low proliferative DPSC (2-10PDs) were accompanied by increased oxidative damage, absent in high proliferative DPSCs until 45-60PDs. Increased SOD2/glutathione S-transferase ζ1 (GSTZ1) expression and SOD activities were identified in high proliferative DPSCs (10-25PDs), which declined during expansion. Low proliferative DPSCs (2-10PDs) exhibited inferior SOD, catalase and glutathione-related antioxidant expression/activities. CONCLUSIONS: Significant variations exist in the susceptibilities of DPSC sub-populations to oxidative damage and premature senescence, contributed to by differential SOD2 and GSTZ1 profiles which maintain senescence-resistance/stemness properties in high proliferative DPSCs. Identification of superior antioxidant properties in high proliferative DPSCs enhances our understanding of DPSC biology and senescence, which may be exploited for selective sub-population screening/isolation from dental pulp tissues for regenerative medicine-based applications.

A New Look at the Purported Health Benefits of Commercial and Natural Clays.

Clays attributed to have medicinal properties have been used since prehistoric times and are still used today as complementary medicines, which has given rise to unregulated "bioceutical" clays to treat skin conditions. Recently, clays with antibacterial characteristics have been proposed as alternatives to antibiotics, potentially overcoming modern day antibiotic resistance. Clays with suggested antibacterial properties were examined to establish their effects on common wound-infecting bacteria. Geochemical, microscopical, and toxicological characterization of clay particulates, their suspensions and filtered leachates was performed on THP-1 and HaCaT cell lines. Cytoskeletal toxicity, cell proliferation/viability (MTT assays), and migration (scratch wounds) were further evaluated. Clays were assayed for antibacterial efficacy using minimum inhibitory concentration assays. All clays possessed a mineral content with antibacterial potential; however, clay leachates contained insufficient ions to have any antibacterial effects. All clay leachates displayed toxicity towards THP-1 monocytes, while clay suspensions showed less toxicity, suggesting immunogenicity. Reduced clay cytotoxicity on HaCaTs was shown, as many leachates stimulated wound-healing responses. The "Green" clay exhibited antibacterial effects and only in suspension, which was lost upon neutralization. pH and its interaction with clay particle surface charge is more significant than previously understood to emphasize dangers of unregulated marketing and unsubstantiated bioceutical claims.

Evaluation of Dental Pulp Stem Cell Heterogeneity and Behaviour in 3D Type I Collagen Gels.

Dental pulp stem cells (DPSCs) are increasingly being advocated for regenerative medicine-based therapies. However, significant heterogeneity in the genotypic/phenotypic properties of DPSC subpopulations exist, influencing their therapeutic potentials. As most studies have established DPSC heterogeneity using 2D culture approaches, we investigated whether heterogeneous DPSC proliferative and contraction/remodelling capabilities were further evident within 3D type I collagen gels in vitro. DPSC subpopulations were isolated from human third molars and identified as high/low proliferative and multipotent/unipotent, following in vitro culture expansion and population doubling (PD) analysis. High proliferative/multipotent DPSCs, such as A3 (30 PDs and 80 PDs), and low proliferative/unipotent DPSCs, such as A1 (17 PDs), were cultured in collagen gels for 12 days, either attached or detached from the surrounding culture plastic. Collagen architecture and high proliferative/multipotent DPSC morphologies were visualised by Scanning Electron Microscopy and FITC-phalloidin/Fluorescence Microscopy. DPSC proliferation (cell counts), contraction (% diameter reductions), and remodelling (MMP-2/MMP-9 gelatin zymography) of collagen gels were also evaluated. Unexpectedly, no proliferation differences existed between DPSCs, A3 (30 PDs) and A1 (17 PDs), although A3 (80 PDs) responses were significantly reduced. Despite rapid detached collagen gel contraction with A3 (30 PDs), similar contraction rates were determined with A1 (17 PDs), although A3 (80 PDs) contraction was significantly impaired. Gel contraction correlated to distinct gelatinase profiles. A3 (30 PDs) possessed superior MMP-9 and comparable MMP-2 activities to A1 (17 PDs), whereas A3 (80 PDs) had significantly reduced MMP-2/MMP-9. High proliferative/multipotent DPSCs, A3 (30 PDs), further exhibited fibroblast-like morphologies becoming polygonal within attached gels, whilst losing cytoskeletal organization and fibroblastic morphologies in detached gels. This study demonstrates that heterogeneity exists in the gel contraction and MMP expression/activity capabilities of DPSCs, potentially reflecting differences in their abilities to degrade biomaterial scaffolds and regulate cellular functions in 3D environments and their regenerative properties overall. Thus, such findings enhance our understanding of the molecular and phenotypic characteristics associated with high proliferative/multipotent DPSCs.

Evaluation of the In Vitro Oral Wound Healing Effects of Pomegranate (Punica granatum) Rind Extract and Punicalagin, in Combination with Zn (II).

Pomegranate (Punica granatum) is a well-established folklore medicine, demonstrating benefits in treating numerous conditions partly due to its antimicrobial and anti-inflammatory properties. Such desirable medicinal capabilities are attributed to a high hydrolysable tannin content, especially punicalagin. However, few studies have evaluated the abilities of pomegranate to promote oral healing, during situations such as periodontal disease or trauma. Therefore, this study evaluated the antioxidant and in vitro gingival wound healing effects of pomegranate rind extract (PRE) and punicalagin, alone and in combination with Zn (II). In vitro antioxidant activities were studied using DPPH and ABTS assays, with total PRE phenolic content measured by Folin-Ciocalteu assay. PRE, punicalagin and Zn (II) combination effects on human gingival fibroblast viability/proliferation and migration were investigated by MTT assay and scratch wounds, respectively. Punicalagin demonstrated superior antioxidant capacities to PRE, although Zn (II) exerted no additional influences. PRE, punicalagin and Zn (II) reduced gingival fibroblast viability and migration at high concentrations, but retained viability at lower concentrations without Zn (II). Fibroblast speed and distance travelled during migration were also enhanced by punicalagin with Zn (II) at low concentrations. Therefore, punicalagin in combination with Zn (II) may promote certain anti-inflammatory and fibroblast responses to aid oral healing.

Novel epoxy-tiglianes stimulate skin keratinocyte wound healing responses and re-epithelialization via protein kinase C activation.

Epoxy-tiglianes are a novel class of diterpene esters. The prototype epoxy-tigliane, EBC-46 (tigilanol tiglate), possesses potent anti-cancer properties and is currently in clinical development as a local treatment for human and veterinary cutaneous tumors. EBC-46 rapidly destroys treated tumors and consistently promotes wound re-epithelialization at sites of tumor destruction. However, the mechanisms underlying these keratinocyte wound healing responses are not completely understood. Here, we investigated the effects of EBC-46 and an analogue (EBC-211) at 1.51 nM-151 µM concentrations, on wound healing responses in immortalized human skin keratinocytes (HaCaTs). Both EBC-46 and EBC-211 (1.51 nM-15.1 µM) accelerated G0/G1-S and S-G2/M cell cycle transitions and HaCaT proliferation. EBC-46 (1.51-151 nM) and EBC-211 (1.51 nM-15.1 µM) further induced significant HaCaT migration and scratch wound repopulation. Stimulated migration/wound repopulation responses were even induced by EBC-46 (1.51 nM) and EBC-211 (1.51-151 nM) with proliferation inhibitor, mitomycin C (1 µM), suggesting that epoxy-tiglianes can promote migration and wound repopulation independently of proliferation. Expression profiling analyses showed that epoxy-tiglianes modulated keratin, DNA synthesis/replication, cell cycle/proliferation, motility/migration, differentiation, matrix metalloproteinase (MMP) and cytokine/chemokine gene expression, to facilitate enhanced responses. Although epoxy-tiglianes down-regulated established cytokine and chemokine agonists of keratinocyte proliferation and migration, enhanced HaCaT responses were demonstrated to be mediated via protein kinase C (PKC) phosphorylation and significantly abrogated by pan-PKC inhibitor, bisindolylmaleimide-1 (BIM-1, 1 µM). By identifying how epoxy-tiglianes stimulate keratinocyte healing responses and re-epithelialization in treated skin, our findings support the further development of this class of small molecules as potential therapeutics for other clinical situations associated with impaired re-epithelialization, such as non-healing skin wounds.

Evaluation of Cypholophus macrocephalus sap as a treatment for infected cutaneous ulcers in Papua New Guinea.

Cypholophus macrocephalus sap is used to treat bacterially infected cutaneous leg ulcers in Papua New Guinea. High resolution LC-MS analysis of the sap revealed it to be rich in sulphated flavonoids. We assessed the effects of the sap on the differentiation and pro-inflammatory anti-microbial responses of M1 macrophages using IL-6 and TNF-α ELISAs and found significant increases in M1 macrophage IL-6 expression with concentrations as low as 243 ng/ml sap. Neutrophil IL-6 and TNF-α expression was also significantly increased but to a lesser degree. Matrix metalloproteinases (MMPs) 1, 2, 8 and 9 which are known to contribute to the toxic nature of wound exudates were inhibited by the sap at 24 µg/ml. The sap was tested with several bacterial species known to colonize cutaneous ulcers in Papua New Guinea but proved not to be active. Cypholophus sap stimulates pro-inflammatory, anti-microbial M1 macrophage and neutrophil responses at very low concentrations, whilst also inhibiting MMPs. The combination of an enhanced innate immune response and inhibition of MMPs in ulcer exudate, may contribute to the eradication of bacteria and healing of these infected ulcers. The sap concentrations used in these assays are readily achievable in an in vivo context.