Determining the potential use of biosurfactants in preventing endodontic infections.

Microbial biofilms play a dominant role in the failure of endodontic therapies. Bacterial adhesion is the first step in the establishment of biofilms, activating the host immune response leading to tissue damage. Biosurfactants are microbe-derived tensioactive molecules with latent anti-adhesive and anti-microbial activity. This study reports the extraction and characterization of a biosurfactant from Lactobacillus (L.) plantarum (Lp-BS) and investigates its anti-microbial and anti-adhesive properties compared to rhamnolipid, a commercially available biosurfactant. Lp-BS, extracted from L. plantarum during the growth phase, was characterized as a glycoprotein, able to reduce surface tension and emulsify non-polar liquids. Proteomic analysis of Lp-BS identified three bacterial adhesin-like proteins, suggesting roles in hindering bacterial adhesion. Lp-BS did not show significant anti-microbial activity against endodontic pathogens from the Streptococcus (Strep.) anginosus group or Enterococcus (Ent.) faecalis at 50 mg/ml. However, anti-adhesive activity on abiotic surfaces was observed against both Strep. anginosus and Strep. intermedius. Rhamnolipid exhibited strong anti-microbial activity, with minimum inhibitory concentrations of 0.097 mg/ml against Strep. anginosus, and 0.048 mg/ml against Strep. constellatus and Strep. intermedius, in addition to a marked anti-adhesive activity. These findings offer preliminary evidence for the potential application of biosurfactants as an anti-microbial and/or anti-adhesive pharmacotherapy in endodontics.

Disrupted circadian clocks and altered tissue mechanics in primary human breast tumours.

BACKGROUND: Circadian rhythms maintain tissue homeostasis during the 24-h day-night cycle. Cell-autonomous circadian clocks play fundamental roles in cell division, DNA damage responses and metabolism. Circadian disruptions have been proposed as a contributing factor for cancer initiation and progression, although definitive evidence for altered molecular circadian clocks in cancer is still lacking. In this study, we looked at circadian clocks in breast cancer. METHODS: We isolated primary tumours and normal tissues from the same individuals who had developed breast cancer with no metastases. We assessed circadian clocks within primary cells of the patients by lentiviral expression of circadian reporters, and the levels of clock genes in tissues by qPCR. We histologically examined collagen organisation within the normal and tumour tissue areas, and probed the stiffness of the stroma adjacent to normal and tumour epithelium using atomic force microscopy. RESULTS: Epithelial ducts were disorganised within the tumour areas. Circadian clocks were altered in cultured tumour cells. Tumour regions were surrounded by stroma with an altered collagen organisation and increased stiffness. Levels of Bmal1 messenger RNA (mRNA) were significantly altered in the tumours in comparison to normal epithelia. CONCLUSION: Circadian rhythms are suppressed in breast tumour epithelia in comparison to the normal epithelia in paired patient samples. This correlates with increased tissue stiffness around the tumour region. We suggest possible involvement of altered circadian clocks in the development and progression of breast cancer.

Variation in human dental pulp stem cell ageing profiles reflect contrasting proliferative and regenerative capabilities.

BACKGROUND: Dental pulp stem cells (DPSCs) are increasingly being recognized as a viable cell source for regenerative medicine. Although significant variations in their ex vivo expansion are well-established, DPSC proliferative heterogeneity remains poorly understood, despite such characteristics influencing their regenerative and therapeutic potential. This study assessed clonal human DPSC regenerative potential and the impact of cellular senescence on these responses, to better understand DPSC functional behaviour. RESULTS: All DPSCs were negative for hTERT. Whilst one DPSC population reached >80 PDs before senescence, other populations only achieved <40 PDs, correlating with DPSCs with high proliferative capacities possessing longer telomeres (18.9 kb) than less proliferative populations (5-13 kb). High proliferative capacity DPSCs exhibited prolonged stem cell marker expression, but lacked CD271. Early-onset senescence, stem cell marker loss and positive CD271 expression in DPSCs with low proliferative capacities were associated with impaired osteogenic and chondrogenic differentiation, favouring adipogenesis. DPSCs with high proliferative capacities only demonstrated impaired differentiation following prolonged expansion (>60 PDs). CONCLUSIONS: This study has identified that proliferative and regenerative heterogeneity is related to contrasting telomere lengths and CD271 expression between DPSC populations. These characteristics may ultimately be used to selectively screen and isolate high proliferative capacity/multi-potent DPSCs for regenerative medicine exploitation.

A 3D ex vivo mandible slice system for longitudinal culturing of transplanted dental pulp progenitor cells.

Harnessing mesenchymal stem cells for tissue repair underpins regenerative medicine. However, how the 3D tissue matrix maintains such cells in a quiescent state whilst at the same time primed to respond to tissue damage remains relatively unknown. Developing more physiologically relevant 3D models would allow us to better understand the matrix drivers and influence on cell-lineage differentiation in situ. In this study, we have developed an ex vivo organotypic rat mandible slice model; a technically defined platform for the culture and characterization of dental pulp progenitor cells expressing GFP driven by the ß-actin promoter (cGFP DPPCs). Using confocal microscopy we have characterized how the native environment influences the progenitor cells transplanted into the dental pulp. Injected cGFP-DPPCs were highly viable and furthermore differentially proliferated in unique regions of the mandible slice; in the dentine region, cGFP-DPPCs showed a columnar morphology indicative of expansion and lineage differentiation. Hence, we demonstrated the systematic capacity for establishing a dental pulp cell-micro-community, phenotypically modified in the tooth (the "biology"); and at the same time addressed technical challenges enabling the mandible slice to be accessible on platforms for high-content imaging (the biology in a "multiplex" format).

Quantification of clonal heterogeneity of mesenchymal progenitor cells in dental pulp and bone marrow.

This study aimed to compare the expression of "classical" stem cell markers, the proliferative capacity and differentiation ability of clonal mesenchymal stem cell (MSC) populations isolated from animal matched dental pulp (DP) and bone marrow (BM) of rats. MSCs were derived from the aforementioned tissues, with immature MSCs selected for by preferential fibronectin-adherence and resultant single-cell derived clonal populations culture expanded. Colony forming efficiencies were 12 times greater for DP clones compared with BM clones. Expansion of isolated colonies, however, was 5 times more successful for BM clones. All clones exceeded 40 population doublings (PDs) and all exhibited periods of high and low proliferative rates. PDs were approximately 1.5 times higher for BM clones. All BM clones readily differentiated towards osteoblasts, chondrocytes and adipocytes. Of the three DP clones analysed, all demonstrated osteogenesis, albeit with reduced efficiency compared to BM clones. One clone demonstrated adipogenesis and one clone chodrogenesis. qPCR determined quantifiable differences in Msx2, Vcam2 and Mcam with no clone showing similarity to another. The expression of a specific mesenchymal marker did not predict proliferative or differentiation potential. These results also suggest lineage restriction of the DP clones.

Differential influence of fluoride concentration on the synthesis of bone matrix glycoproteins within mineralizing bone cells in vitro.

OBJECTIVE: This study investigated the influence of fluoride levels on the temporal synthesis of bone-associated glycoproteins, which have been assigned prominent roles in regulating crystal growth, size and shape during the mineralization process. MATERIALS AND METHODS: Bone marrow stromal cells were isolated from male Wistar rats and cultured under mineralizing conditions, supplemented with 0 M, 10(-7) M or 10(-5) M sodium fluoride. The presence of bone-associated glycoproteins was examined 2-13 days post-reseeding by immunocytochemical localization. Results: All bone-associated glycoproteins increased in 10(-7) M fluoride, compared to untreated controls, particularly at days 6 and 13 in culture. Conversely, higher 10(-5) M fluoride concentrations decreased glycoprotein levels, compared to controls. CONCLUSIONS: Results highlight a differential effect of fluoride concentration on glycoprotein synthesis by osteoblasts.

Nanoparticle and Nanotopography-Induced Activation of the Wnt Pathway in Bone Regeneration.

Background and Aims: Recent research has focused on developing nanoparticle and nanotopography-based technologies for bone regeneration. The Wingless-related integration site (Wnt) signaling pathway has been shown to play a vital role in this process, in particular in osteogenic differentiation and proliferation. The exact mechanisms by which nanoparticles and nanotopographies activate the Wnt signaling pathway, however, are not fully understood. This review aimed to elucidate the mechanisms by which nanoscale technologies activate the Wnt signaling pathway during bone regeneration. Methods: The terms "Wnt," "bone," and "nano*" were searched on PubMed and Ovid with no date limit. Only original research articles related to Wnt signaling and bone regeneration in the context of nanotopographies, nanoparticles, or scaffolds with nanotopographies/nanoparticles were reviewed. Results: The primary mechanism by which nanoparticles activated the Wnt pathway was by internalization through the endocytic pathway or diffusion through the cell membrane, leading to accumulation of nonphosphorylated ß-catenin in the cytoplasm and subsequently downstream osteogenic signaling (e.g., upregulation of runt-related transcription factor 2 [RUNX2]). The specific size of the nanoparticles and the process of endocytosis itself has been shown to modulate the Wnt-ß-catenin pathway. Nanotopographies were shown to directly activate frizzled receptors, initiating Wnt/ß-catenin signaling. Additional studies showed nanotopographies to activate the Wnt/calcium (Wnt/Ca2+)-dependent and Wnt/planar cell polarity pathways through nuclear factor of activated T cells, and α5ß1 integrin stimulation. Finally, scaffolds containing nanotopographies/nanoparticles were found to induce Wnt signaling through a combination of ion release (e.g., lithium, boron, lanthanum, and icariin), which inhibited glycogen synthase kinase 3 beta (GSK-3ß) activity, and through similar mechanisms to the nanotopographies. Conclusion: This review concludes that nanoparticles and nanotopographies cause Wnt activation through several different mechanisms, specific to the size, shape, and structure of the nanoparticles or nanotopographies. Endocytosis-related mechanisms, integrin signaling and ion release were the major mechanisms identified across nanoparticles, nanotopographies, and scaffolds, respectively. Knowledge of these mechanisms will help develop more effective targeted nanoscale technologies for bone regeneration. Impact statement Nanoparticles and nanotopographies can activate the Wingless-related integration site (Wnt) signaling pathway, which is essential for bone regeneration. This review has identified that activation is due to endocytosis, integrin signaling and ion release, depending on the size, shape, and structure of the nanoparticles or nanotopographies. By identifying and further understanding these mechanisms, more effective nanoscale technologies that target the Wnt signaling pathway can be developed. These technologies can be used for the treatment of nonunion bone fractures, a major clinical challenge, with the potential to improve the quality of life of millions of patients around the world.

Situational and Dispositional Factors in Rape Cognitions: The Roles of Social Media and the Dark Triad Traits.

Previous research has established the importance of socially aversive personality traits (i.e., the Dark Triad) in rape cognitions (operationalized here as rape-supportive attitudes, rape victim empathy, and hostile masculinity). However, less is known about how sexist social media content influences attitudes toward rape cognitions depending on the personality of the individual. In an online experiment, after completing the Short Dark Triad-3 questionnaire, participants (N = 180) were primed with either sexist or neutral tweets, rating them for acceptability, humor, rudeness, and ignorance. Participants then completed scales for rape-supportive attitudes, victim empathy, and hostile masculinity. Sexist tweets were rated as significantly less acceptable and humorous, and more rude and ignorant than neutral tweets. However, those high in the Dark Triad found the sexist tweets as funny and acceptable. Overall, exposure to the sexist tweets did not increase rape cognitions. Moreover, the Dark Triad traits had similar significant, positive correlations with rape-supportive attitudes, victim blame, and hostile masculinity in both sexist and neutral tweet conditions. Multiple regression analyses (controlling for gender) revealed that psychopathy was the strongest positive predictor for increased rape cognitions. Findings suggest that short exposure to sexist social media content may not influence rape cognitions, but that dispositional factors such as psychopathy are more important.

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.

Characterization of oxidative stress status during diabetic bone healing.

Early events associated with bone healing in patients with type 2 diabetes mellitus appear to be delayed. Hyperglycaemia and an associated increase in oxidative stress are cited as potential factors leading to a change in cellular behaviour. Using an in vivo model monitoring bone formation around implants placed into rat mandibles, we have previously identified that the onset of cell proliferation and osteoblast differentiation are delayed and subsequently prolonged compared with normal bone. This study used the same implant model to characterize oxidative stress biomarkers and primary antioxidant enzyme profiles during diabetic bone healing in vivo. Implants were placed into the sockets of incisors extracted from the mandibles of normal Wistar and diabetic Goto-Kakizaki rats for 3 and 9 weeks after implant insertion. Histochemical analysis confirmed a delay in bone healing around implants in diabetic animals. Immunohistochemical localization of peri-cellular staining for protein carbonyl groups, as a biomarker of oxidized protein content, was slightly higher in diabetic granulation tissue compared with normal tissue. However, no differences were observed in the staining patterns of advanced glycation end products. Minimal differences were observed in the number of cells positive for cytoplasmic superoxide dismutase (SOD)1 or mitochondrial SOD2. Significantly, catalase was absent in diabetic tissues. The results suggest that the oxidative environment in healing bone is differentially affected by hyperglycaemia, particularly in relation to catalase. The significance of these observations for diabetic bone healing is discussed.

Cooperative spin transition in the two-dimensional coordination polymer [Fe(4,4'-bipyridine)2(NCX)2]·4CHCl3 (X = S, Se).

Two new isostructural two-dimensional (2D) coordination polymers exhibiting spin crossover (SCO) behavior of formulation [Fe(4,4'-bipy)(2)(NCX)(2)]·4CHCl(3) (4,4'-bipy = 4,4'-bipyridine; X = S [1·4CHCl(3)], Se [2·4CHCl(3)]) have been synthesized and characterized, and both undergo cooperative spin transitions (ST). For 1·4CHCl(3) the ST takes place in two steps with critical temperatures of T(c1)(down) = 143.1 K, T(c2)(down) = 91.2 K, T(c1)(up) = 150.7 K, and T(c2)(up) = 112.2 K. 2·4CHCl(3) displays half ST characterized by T(c)(down) = 161.7 K and T(c)(up) = 168.3 K. The average enthalpy and entropy variations and cooperativity parameters associated with the ST have been estimated to be ΔH(1)(av) = 5.18 kJ mol(-1), ΔS(1)(av) = 35 J K(-1) mol(-1), and Γ(1) = 2.8 kJ mol(-1) and ΔH(2)(av) = 3.55 kJ mol(-1), ΔS(2)(av) = 35 J K(-1) mol(-1), and Γ(2) = 2.6 kJ mol(-1) for 1·4CHCl(3), and ΔH(av) = 6.25 kJ mol(-1), ΔS(av) = 38.1 J K(-1) mol(-1), and Γ = 3.2 kJ mol(-1) for 2·4CHCl(3). At T > [T(c1) (1·4CHCl(3)); T(c) (2·4CHCl(3))], both compounds are in the space group P2/c while at T < [T(c1) (1·4CHCl(3)); T(c) (2·4CHCl(3))] they change to the C2/c space group and display an ordered checkerboard-like arrangement of iron(II) sites where the high- and low-spin states coexist at 50%.

Delayed osteoblast differentiation and altered inflammatory response around implants placed in incisor sockets of type 2 diabetic rats.

OBJECTIVE: Central to the process of osseointegration is the recruitment of mesenchymal progenitor cells to the healing site, their proliferation and differentiation to bone synthesising osteoblasts. The process is under the control of pro-inflammatory cytokines and growth factors. The aim of this study was to monitor these key stages of osseointegration and the signalling milieu during bone healing around implants placed in healthy and diabetic bone. METHODS: Implants were placed into the sockets of incisors extracted from the mandibles of normal Wistar and diabetic Goto-Kakizaki rats. Mandibles 1-12 weeks post-insertion of the implant were examined by histochemistry and immunocytochemistry to localise the presence of Stro-1- positive mesenchymal progenitor cells, proliferating cellular nuclear antigen proliferative cells, osteopontin and osteocalcin, macrophages, pro-inflammatory cytokines interleukin (IL)-1ß, IL-6, tumour necrosis factor (TNF)-α and tumour growth factor (TGF)-ß1. Image analysis provided a semi-quantification of positively expressing cells. RESULTS: Histological staining identified a delay in the formation of mineralised bone around implants placed in diabetic animals. Within the diabetic bone, the migration of Stro-1 mesenchymal cells in the healing tissue appeared to be unaffected. However, in the diabetic healing bone, the onset of cell proliferation and osteoblast differentiation were delayed and subsequently prolonged compared with normal bone. Similar patterns of change were observed in diabetic bone for the presence of IL-1ß, TNF-α, macrophages and TGF-ß1. CONCLUSION: The observed alterations in the extracellular presence of pro-inflammatory cytokines, macrophages and growth factors within diabetic tissues that correlate to changes in the signalling milieu, may affect the proliferation and differentiation of mesenchymal progenitor cells in the osseointegration process.

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.

Exploring a Chemotactic Role for EVs from Progenitor Cell Populations of Human Exfoliated Deciduous Teeth for Promoting Migration of Naïve BMSCs in Bone Repair Process.

Mobilization of naïve bone marrow mesenchymal stromal cells (BMSCs) is crucial to desired bone regeneration in both orthopedic and dental contexts. In such conditions, mesenchymal progenitor cell populations from human exfoliated deciduous teeth (SHEDs) present advantageous multipotent properties with easy accessibility which makes them a good candidate in both bone and periodontal tissue regeneration. Extracellular vesicles (EVs) are a functional membranous structure which could participate in multiple cell interactions and imitate the biological functions of their parenting cells largely. To assess their ability to mobilize naïve BMSCs in the bone repair process, Nanosight Tracking Analysis (NTA) and Enzyme-Linked Immunosorbent Assays (ELISA) were performed to illustrate the composition and functional contents of EV samples derived from SHEDs with different culturing time (24 h, 48 h, and 72 h). Afterwards, the Boyden chamber assay was performed to compare their capacity for mobilizing naïve BMSCs. One-way analysis of variance (ANOVA) with a post hoc Turkey test was performed for statistical analysis. SHEDs-derived EVs collected from 24 h, 48 h, and 72 h time points, namely, EV24, EV48, and EV72, were mainly secreted as exosomes and tended to reform into smaller size as a result of sonication indicated by NTA results. Moreover, different EV groups were found to be abundant with multiple growth factors including transforming growth factor-ß1 (TGF-ß1), platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), and fibroblast growth factor-2 (FGF-2) given the detections through ELISA. Boyden chamber assays implied the migratory efficiency of BMSCs driven by EVs at varying concentrations. However, the results showed that migration of BMSCs driven by different EV groups was not statistically significant even with chemotactic factors contained (P > 0.05). Taken together, these data suggest that EVs derived from SHEDs are secreted in functional forms and present a potential of mobilizing naïve BMSCs, which may propose their relevance in assisting bone regeneration.

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.

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.

Interrogating the Osteogenic Potential of Implant Surfaces In Vitro: A Review of Current Assays.

The success of implantable devices relies heavily on their interaction with the host cells facilitating the osseointegration process. However, with so many new surface modifications, with subtly varying design parameters, in vitro assays can, with proper interpretation, provide valuable information for understanding cellular behavior. This review brings together pertinent in vitro experimental protocols available to researchers and discusses them in relationship to the development of the osteoblast phenotype during bone repair. Consideration is also paid to the influence of endothelial and macrophage cells that can substantially change osteogenic cell activity and thus can provide added value for predicting the osseointegration potential in vivo. Due to the diverse and heterogeneous nature of cell types available for culture use, this review concludes that there is no "gold standard" series of assays. Rather, we present guidance in the experimental design of in vitro assays to better identify those surfaces with promising osteogenic potential. Impact statement Titanium implants are already widely used in orthopedics and dentistry, yet, intensive research continues with the aim of modifying and functionalizing implant surfaces to invoke a stronger bone response and to meet current clinical challenges around improving longevity, decreasing morbidity, widening access, and clinical application. A very large number of surface modifications have been studied and the potential for new designs appears to be limitless as new technology grows. This review provides guidance for in vitro assays available to test these technologies, providing a cost-effective means for acquiring robust and physiologically relevant data, before in vivo examination.

Isolation of distinct progenitor stem cell populations from dental pulp.

The present study compared the cellular characteristics of progenitor stem cell populations present in adult dental pulp, isolated by different methods utilizing 2 different features of stem cell biology. One population expressing high levels of beta1 integrin was isolated by preferential selection of adherent cells to fibronectin over 20 min. In an alternative approach, cells expressing the embryonic neural crest cell marker, low-affinity nerve growth factor receptor (LANGFR), were selected by magnetic-activated cell sorting. For each method, clonal cell lines were established and expanded in culture. One clone derived via the respective methods was examined for embryonic/progenitor cell markers by immunocytochemistry and RT-PCR. Both clonal populations demonstrated the expression of stro-1 and stained positive for vimentin, demonstrating mesenchymal lineage. Of note, cells selected for LANGFR cells demonstrated the additional expression of CD105 and Notch 2. For both clonal populations, expanded cultures demonstrated the ability to differentiate into osteoblasts, adipocytes and chondrocytes. These results would suggest the potential isolation of 2 progenitor cell populations exhibiting different cellular characteristics in terms of their embryonic nature. The potential for both cell populations to derive from a common origin is discussed.

Dental pulp stem cells: what, where, how?

INTRODUCTION: It is now accepted that progenitor/stem cells reside within the post-natal dental pulp. Studies have identified several niches of multipotent mesenchymal progenitor cells, known as dental pulp stem cells, which have a high proliferative potential for self-renewal. These progenitor stem cells are now recognized as being vital to the dentine regeneration process following injury. Understanding the nature of these progenitor/stem cell populations in the pulp is important in determining their potentialities and development of isolation or recruitment strategies for use in regeneration and tissue engineering. Characterization of these cells, and determination of their potentialities in terms of specificity of regenerative response, may help direct new clinical treatment modalities. Such novel treatments may involve controlled direct recruitment of the cells in situ and possible seeding of stem cells at sites of injury for regeneration or use of the stem cells with appropriate scaffolds for tissue engineering solutions. Such approaches may provide an innovative and novel biologically based new generation of clinical materials and/or treatments for dental disease. AIM: This study aimed to review the body of knowledge relating to stem cells and to consider the possibility of these cell populations, and related technology, in future clinical applications.

Adverse Childhood Experiences and Psychological Distress in Juvenile Offenders: The Protective Influence of Resilience and Youth Assets.

PURPOSE: To examine whether internal resiliency and external assets directly protect juvenile offenders exposed to adverse childhood experiences (ACEs) from psychological distress and moderate the relationship between ACE exposure and psychological distress. METHODS: A total of 429 male and female adolescents involved with juvenile justice systems in a Western state completed an audio computer-assisted self-interview. Validated measures assessed ACEs, psychological distress, internal resiliency, and external youth assets. Hierarchical linear regression was used to assess the direct and moderating protective effects of internal resilience, family communication, school connectedness, peer role models, and nonparental role models on psychological distress. All models controlled for age, sex, race/ethnicity, free/reduced lunch qualification, current custody, supervision status, detention, and site. RESULTS: The mean ACE score among participants was 3.7 (standard deviation = 2.2) and 52.8% reported four or more ACEs. Participants with 4-5 ACEs (ß = .37, p < .001) and 6-8 ACEs (ß = .49, p < .001) were at increased risk for psychological distress. High internal resilience (ß = -.20, p < .001), family communication (ß = -.19, p < .001), school connectedness (ß = -.14, p < .01), and peer role models (ß = -.09, p < .05) were associated with a reduction in psychological distress in the presence of high ACE exposure. In the interaction models, having a high number of ACEs remained strongly associated with increased psychological distress. However, internal resilience (ß = -.24, p < .01) and school connectedness (ß = -.18, p < .05) significantly moderated (reduced) the relationship between high ACE exposure and psychological distress. CONCLUSIONS: Our findings suggest that programs and policies that promote internal resilience and protective factors across multiple levels of influence may protect juvenile offenders exposed to childhood trauma from psychological distress.