Primary care dentistry: An Australian perspective.

INTRODUCTION: Primary care dentistry is the first point of contact that someone has with the dental system and is predominantly focused on the treatment and prevention of dental caries and periodontal disease. The aim of this paper was to review the Australian primary dental care system. METHODS: This paper reviews the primary dental care system in Australia, drawing on data reporting on the dental workforce, funding sources for dental care, oral health outcome measures and dental visiting patterns. RESULTS: Primary dental health care in Australia is predominantly provided by dentists working in private practice, with the number of dentists per 100,000 people in Australia increasing from 46.9 in 2000 to 65.1 in 2022. However, there has been a gradual shift over the past twenty years towards greater service provision by other members of the dental team who now represent one quarter of the dental workforce, and some expansion of publicly funded dental care. Despite this dentistry remains isolated from the rest of primary health care, and the lack of government funding means that many people continue to miss out of necessary dental care, particularly those living in regional and rural Australia and from low-income groups. CONCLUSIONS: Australians should be able to access primary dental care services when and where they need it with adequate financial protection, from services that are well integrated into the broader primary health care system to ensure they are able to achieve optimal oral and general health. For many Australians, this is not currently the case. CLINICAL SIGNIFICANCE: Australia is at a crossroads with respect to access to dental care, and there is a need for stronger advocacy from stakeholders to improve oral health outcomes and reduce inequalities.

Stem cell sources from human biological waste material: a role for the umbilical cord and dental pulp stem cells for regenerative medicine.

Stem cell research with biological waste material is an area that holds promise to revolutionize treatment modalities and clinical practice. The interest in surgical remnants is increasing with time as research on human embryonic stem cells remains controversial due to legal and ethical issues. Perhaps, these restrictions are the motivation for the use of alternative mesenchymal stem cell (MSC) sources in the regenerative field. Stem cells (SCs) of Umbilical Cord (UC) and Dental Pulp (DP) have almost similar biological characteristics to other MSCs and can differentiate into a number of cell lineages with enormous potential future prospects. A concise critical observation of UC-MSCs and DP-MSCs is presented here reviewing articles from the last two decades along with other stem cell sources from different biological waste materials.

Modulation of MG-63 Osteogenic Response on Mechano-Bactericidal Micronanostructured Titanium Surfaces.

Despite recent advances in the development of orthopedic devices, implant-related failures that occur as a result of poor osseointegration and nosocomial infection are frequent. In this study, we developed a multiscale titanium (Ti) surface topography that promotes both osteogenic and mechano-bactericidal activity using a simple two-step fabrication approach. The response of MG-63 osteoblast-like cells and antibacterial activity toward Pseudomonas aeruginosa and Staphylococcus aureus bacteria was compared for two distinct micronanoarchitectures of differing surface roughness created by acid etching, using either hydrochloric acid (HCl) or sulfuric acid (H2SO4), followed by hydrothermal treatment, henceforth referred to as either MN-HCl or MN-H2SO4. The MN-HCl surfaces were characterized by an average surface microroughness (Sa) of 0.8 ± 0.1 µm covered by blade-like nanosheets of 10 ± 2.1 nm thickness, whereas the MN-H2SO4 surfaces exhibited a greater Sa value of 5.8 ± 0.6 µm, with a network of nanosheets of 20 ± 2.6 nm thickness. Both micronanostructured surfaces promoted enhanced MG-63 attachment and differentiation; however, cell proliferation was only significantly increased on MN-HCl surfaces. In addition, the MN-HCl surface exhibited increased levels of bactericidal activity, with only 0.6% of the P. aeruginosa cells and approximately 5% S. aureus cells remaining viable after 24 h when compared to control surfaces. Thus, we propose the modulation of surface roughness and architecture on the micro- and nanoscale to achieve efficient manipulation of osteogenic cell response combined with mechanical antibacterial activity. The outcomes of this study provide significant insight into the further development of advanced multifunctional orthopedic implant surfaces.

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.

Growth factor release and dental pulp stem cell attachment following dentine conditioning: An in vitro study.

AIM: The aim of the study was to investigate the effect of dentine conditioning agents on growth factor liberation and settlement of dental pulp progenitor cells (DPSCs) on dentine surfaces. METHODOLOGY: The agents used included ethylenediaminetetraacetic acid (EDTA; 10%, pH 7.2), phosphoric acid (37%, pH < 1), citric acid (10%, pH 1.5) and polyacrylic acid (25%, pH 3.9). Human dentine slices were conditioned for exaggerated conditioning times of 5 and 10 min, so that the growth factor liberation reached quantifiable levels above the limit of detection of the laboratory methods employed. Transforming growth factor beta-1 (TGF-ß1) release and surface exposure were quantified by enzyme-linked immunosorbent assay (ELISA) and immunogold labelling. Scanning electron microscopy (SEM) was used to assess the morphology of cells and coverage by DPSCs cultured on dentine surfaces for 8 days. RESULTS: After 5-min conditioning of dentine slices, citric acid was the most effective agent for growth factor release into the aqueous environment as measured by ELISA (Mann-Whitney U with Bonferroni correction, p < .01 compared with phosphoric and polyacrylic acid). As well as this, dentine slices treated with phosphoric acid for the same period, displayed significantly less TGF-ß1 on the surface compared with the other agents used, as measured by immunogold labelling (MWU with Bonferroni correction, p < .05). After 8 days, widespread coverage by DPSCs on dentine surfaces conditioned with citric acid and EDTA were evident under SEM. On dentine surfaces conditioned with phosphoric and polyacrylic acid, respectively, less spread cells and inconsistent cell coverage were observed. CONCLUSIONS: Based on the findings of this in vitro study, a desirable biological growth factor-mediated effect may be gained when conditioning dentine by milder acidic or chelating agents such as citric acid and EDTA. The results must be interpreted in the context that the potential of the applied materials inducing a desirable biological response in DPSCs is only one consideration amongst other important ones in a clinical setting. However, it is crucial to look beyond the mere physical effects of materials and move towards biologically based treatment approaches as far as the restorative management of teeth with viable dental pulps are concerned.

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.

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.

Measuring Antibiotic Stewardship Programmes and Initiatives: An Umbrella Review in Primary Care Medicine and a Systematic Review of Dentistry.

Antibiotic stewardship aims to tackle the global problem of drug-resistant infections by promoting the responsible use of antibiotics. Most antibiotics are prescribed in primary care and widespread overprescribing has been reported, including 80% in dentistry. This review aimed to identify outcomes measured in studies evaluating antibiotic stewardship across primary healthcare. An umbrella review was undertaken across medicine and a systematic review in dentistry. Systematic searches of Ovid Medline, Ovid Embase and Web of Science were undertaken. Two authors independently selected and quality assessed the included studies (using Critical Appraisal Skills Programme for the umbrella review and Quality Assessment Tool for Studies with Diverse Designs for the systematic review). Metrics used to evaluate antibiotic stewardship programmes and interventions were extracted and categorized. Comparisons between medical and dental settings were made. Searches identified 2355 medical and 2704 dental studies. After screening and quality assessment, ten and five studies, respectively, were included. Three outcomes were identified across both medical and dental studies: All focused on antibiotic usage. Four more outcomes were found only in medical studies: these measured patient outcomes, such as adverse effects. To evaluate antibiotic stewardship programmes and interventions across primary healthcare settings, measures of antibiotic use and patient outcomes are recommended.

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.

A core curriculum in the biological and biomedical sciences for dentistry.

INTRODUCTION: The biomedical sciences (BMS) are a central part of the dental curriculum that underpins teaching and clinical practice in all areas of dentistry. Although some specialist groups have proposed curricula in their particular topic areas, there is currently no overarching view of what should be included in a BMS curriculum for undergraduate dental programmes. To address this, the Association for Dental Education in Europe (ADEE) convened a Special Interest Group (SIG) with representatives from across Europe to develop a consensus BMS curriculum for dental programmes. CURRICULUM: This paper summarises the outcome of the deliberations of this SIG and details a consensus view from the SIG of what a BMS curriculum should include. CONCLUSIONS: Given the broad nature of BMS applied to dentistry, this curriculum framework is advisory and seeks to provide programme planners with an indicative list of topics which can be mapped to specific learning objectives within their own curricula. As dentistry becomes increasingly specialised, these will change, or some elements of the undergraduate curriculum may move to the post-graduate setting. So, this document should be seen as a beginning and it will need regular review as BMS curricula in dentistry evolve.

Mucoadhesive thin films for the simultaneous delivery of microbicide and anti-inflammatory drugs in the treatment of periodontal diseases.

There is an unmet clinical need for new products to address the high percentage of the populous who present with periodontal diseases. Drug dose retention at the point of application would facilitate sustained release and more efficacious treatments. The aim of this study was to evaluate mucoadhesive polymeric thin films for simultaneous in situ delivery chlorhexidine and anti-inflammatory and analgesic drugs. Mucoadhesive thin films were prepared using a polymer mixture containing chlorhexidine (25 mg) ± diclofenac sodium (10 and 50 mg), and lidocaine hydrochloride (10 mg) or betamethasone dipropionate (10 and 50 mg). The films were assessed for in vitro drug release and localised tissue delivery, followed by determination of modulated prostaglandin E2 (PGE2) levels in ex vivo tissue and cytotoxicity using a HaCaT keratinocyte cell line. Antibacterial activity of the chlorhexidine/diclofenac film was determined against planktonic and biofilm bacteria associated with periodontal disease and dental plaque. Chlorhexidine release was consistently low (up to 10% of initial loading) from all films, whereas the release of diclofenac, betamethasone and lidocaine exceeded 50% within 30 min. The 50 mg betamethasone film released up to 4-fold more than the 10 mg film. Statistically significant reduction of PGE2 was observed in ex vivo porcine gingival tissue for films containing chlorhexidine with or without diclofenac, and betamethasone. No cytotoxicity was observed for any film, apart from 50 mg betamethasone at 24 h. Films loaded with chlorhexidine and diclofenac were inhibitory against relevant test bacteria. Between 3 and 6 log10 reductions in bacterial cell recovery was observed after biofilm exposure to the chlorhexidine films irrespective of the presence of the anti-inflammatory or anaesthetic. This work demonstrated that thin film formulations have the potential to simultaneously counter key causative factors in periodontal diseases, namely associated bacteria biofilm and chronic local inflammation.

Discrimination of Dental Pulp Stem Cell Regenerative Heterogeneity by Single-Cell Raman Spectroscopy.

IMPACT STATEMENT: This study is the first to investigate and confirm the effectiveness of single-cell Raman spectroscopy (SCRM), in its ability to discriminate between dental pulp stem cells (DPSCs) with contrasting proliferative and differentiation capabilities. The findings show that SCRM can rapidly and noninvasively distinguish and identify DPSC subpopulations in vitro with superior proliferative and multipotency properties, versus lesser quality DPSCs, thereby overcoming the significant heterogeneity issues surrounding DPSC ex vivo expansion and differentiation capabilities. Such findings support further SCRM assessment for the selective screening/isolation of superior quality DPSCs from whole dental pulp tissues, for more effective in vitro evaluation and therapy development.

Efficacy of copolymer scaffolds delivering human demineralised dentine matrix for bone regeneration.

Poly(L-lactide-co-ε-caprolactone) scaffolds were functionalised by 10 or 20 µg/mL of human demineralised dentine matrix. Release kinetics up to 21 days and their osteogenic potential on human bone marrow stromal cells after 7 and 21 days were studied. A total of 390 proteins were identified by mass spectrometry. Bone regeneration proteins showed initial burst of release. Human bone marrow stromal cells were cultured on scaffolds physisorbed with 20 µg/mL and cultured in basal medium (DDM group) or physisorbed and cultured in osteogenic medium or cultured on non-functionalised scaffolds in osteogenic medium. The human bone marrow stromal cells proliferated less in demineralised dentine matrix group and activated ERK/1/2 after both time points. Cells on DDM group showed highest expression of IL-6 and IL-8 at 7 days and expressed higher collagen type 1 alpha 2, SPP1 and bone morphogenetic protein-2 until 21 days. Extracellular protein revealed higher collagen type 1 and bone morphogenetic protein-2 at 21 days in demineralised dentine matrix group. Cells on DDM group showed signs of mineralisation. The functionalised scaffolds were able to stimulate osteogenic differentiation of human bone marrow stromal cells.

Wnt-GSK3ß/ß-Catenin Regulates the Differentiation of Dental Pulp Stem Cells into Bladder Smooth Muscle Cells.

Smooth muscle cell- (SMC-) based tissue engineering provides a promising therapeutic strategy for SMC-related disorders. It has been demonstrated that human dental pulp stem cells (DPSCs) possess the potential to differentiate into mature bladder SMCs by induction with condition medium (CM) from bladder SMC culture, in combination with the transforming growth factor-ß1 (TGF-ß1). However, the molecular mechanism of SMC differentiation from DPSCs has not been fully uncovered. The canonical Wnt signaling (also known as Wnt/ß-catenin) pathway plays an essential role in stem cell fate decision. The aim of this study is to explore the regulation via GSK3ß and associated downstream effectors for SMC differentiation from DPSCs. We characterized one of our DPSC clones with the best proliferation and differentiation abilities. This stem cell clone has shown the capacity to generate a smooth muscle layer-like phenotype after an extended differentiation duration using the SMC induction protocol we established before. We further found that Wnt-GSK3ß/ß-catenin signaling is involved in the process of SMC differentiation from DPSCs, as well as a serial of growth factors, including TGF-ß1, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor-homodimer polypeptide of B chain (BB) (PDGF-BB), and vascular endothelial growth factor (VEGF). Pharmacological inhibition on the canonical Wnt-GSK3ß/ß-catenin pathway significantly downregulated GSK3ß phosphorylation and ß-catenin activation, which in consequence reduced the augmented expression of the growth factors (including TGF-ß1, HGF, PDGF-BB, and VEGF) as well as SMC markers (especially myosin) at a late stage of SMC differentiation. These results suggest that the canonical Wnt-GSK3ß/ß-catenin pathway contributes to DPSC differentiation into mature SMCs through the coordination of different growth factors.

Real-time binding kinetic analyses of the interaction of the dietary stain orange II with dentin matrix.

OBJECTIVES: Dietary stains can be adsorbed into the dentin of teeth. Using Orange II as a model dietary stain, this study investigated the strength of its interaction with the mineral and protein components of dentin matrix and how hydrogen peroxide (H2O2) treatment influences this interaction. METHODS: Dentin slices were prepared from human teeth and were either deproteinized (5.6% sodium hypochlorite, 12 days), demineralised (0.5 M EDTA, 3 days) or left as intact control samples. Samples were stained with Orange II for 1-168 h, during which staining intensity was quantified by image analysis. Similarly, uptake of stain by deproteinized / demineralized samples treated with 10 or 30% H2O2 was investigated. Using surface plasmon resonance technology, real-time binding kinetics were determined assessing the interaction of Orange II with the dentin matrix protein constituents, collagen type I, biglycan, decorin, dentin sialoprotein and osteopontin. RESULTS: Deproteinization of dentin matrix reduced the uptake of the Orange II compared to the intact control. Conversely, demineralization of dentin samples increased the uptake of the dye. Treatment of samples for 48 h with H2O2 reduced subsequent uptake of the Orange II. Real-time kinetic analysis indicated moderate strength of binding for Orange II with collagen type I, weak binding with decorin and biglycan and negligible binding with dentine sialoprotein and osteopontin. CONCLUSION: These results indicate a predominant role for collagen type I, which accounts for 90% of the organic protein matrix of teeth, for attracting dietary stains. Binding analyses indicate that the interaction is highly dissociable, and further binding is reduced following H2O2 treatment. CLINICAL SIGNIFICANCE: This study provides new information regarding adsorption of dietary stains into tooth dentin, suggesting that they are attracted and moderately bound to the collagen type I matrix. This study also contributes valuable information for discussion for considering the effect of H2O2 on bleaching teeth and its influence on subsequent uptake of dietary stains following whitening treatments.

An ex-vivo model to determine dental pulp responses to heat and light-curing of dental restorative materials.

AIM: Based on histological studies from the 1960s, it is recommended that dental pulp temperature increases should not exceed 5.5 °C. However, no contemporary reliable models exist to explore the effects of heat on living dental pulp. The aim of this project was to develop a clinically valid model for studying temperature increases caused by three commonly-used light curing units (LCUs). METHODS: Temperature increases caused by LCUs at varying exposure times and via various thicknesses of dentine were recorded using traditional approaches (i.e. thermocouple device on a laboratory bench) and an ex-vivo tooth slice model. Histomorphometric and immunohistochemical (IL-1ß, HSP70, caspase-3) analysis was performed of the tooth slice model following varying exposure and culture times. RESULTS: Reduced dentine thickness and increased exposure time led to increases in temperature. Whilst the majority of temperature increases recorded using the traditional approach (53 of 60) were greater than the recommended 5.5 °C, 52 of the 60 reference points recorded using the ex-vivo tooth slice model resulted in temperature increases of less than 5.5 °C. Temperature increases of 5.5 °C or more that are prolonged for 40 s caused an immediate decrease in cell number. IL-1ß was not detected in any samples, while HSP70 was detectable immediately after exposure to a temperature increase of 6 °C or more. Higher levels of HSP70 were detected after 24 h culture in tooth slices that experienced a temperature increase of 7.5 °C or more. Low levels of caspase-3 were detected in tooth slices exposed to temperature increase of 7.5 °C or more. CONCLUSION: Experimental arrangements for assessing LCU performance that measure temperature increases using a thermocouple device on a laboratory bench should no longer be used. Future studies in this area should include replication of the clinical environment using greater sophistication, such as the use of an ex-vivo tooth slice model as described here. Temperature increases of 5.5 °C or more for 40 s caused an immediate decrease in cell number, which supports previous findings. However, complex interactions at an immunohistochemical level suggest that while temperature increases of 5 °C or less are ideal, there may be some cell damage between 5-7 °C which might not result in pulpal death. Further investigations are indicated.

Isolation and Characterisation of Mesenchymal Stem Cells from Rat Bone Marrow and the Endosteal Niche: A Comparative Study.

Within bone, mesenchymal stromal cells (MSCs) exist within the bone marrow stroma (BM-MSC) and the endosteal niche, as cells lining compact bone (CB-MSCs). This study isolated and characterised heterogeneous MSC populations from each niche and subsequently investigated the effects of extensive cell expansion, analysing population doublings (PDs)/cellular senescence, colony-forming efficiencies (CFEs), MSC cell marker expression, and osteogenic/adipogenic differentiation. CB-MSCs and BM-MSCs demonstrated similar morphologies and PDs, reaching 100 PDs. Both populations exhibited consistent telomere lengths (12-17 kb), minimal senescence, and positive telomerase expression. CB-MSCs (PD15) had significantly lower CFEs than PD50. CB-MSCs and BM-MSCs both expressed MSC (CD73/CD90/CD105); embryonic (Nanog) and osteogenic markers (Runx2, osteocalcin) but no hematopoietic markers (CD45). CB-MSCs (PD15) strongly expressed Oct4 and p16INK4A. At early PDs, CB-MSCs possessed a strong osteogenic potency and low potency for adipogenesis, whilst BM-MSCs possessed greater overall bipotentiality for osteogenesis and adipogenesis. At PD50, CB-MSCs demonstrated reduced potency for both osteogenesis and adipogenesis, compared to BM-MSCs at equivalent PDs. This study demonstrates similarities in proliferative and mesenchymal cell characteristics between CB-MSCs and BM-MSCs, but contrasting multipotentiality. Such findings support further comparisons of human CB-MSCs and BM-MSCs, facilitating selection of optimal MSC populations for regenerative medicine purposes.