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.

Electric signals counterbalanced posterior vs anterior PTEN signaling in directed migration of Dictyostelium.

BACKGROUND: Cells show directed migration response to electric signals, namely electrotaxis or galvanotaxis. PI3K and PTEN jointly play counterbalancing roles in this event via a bilateral regulation of PIP3 signaling. PI3K has been proved essential in anterior signaling of electrotaxing cells, whilst the role of PTEN remains elusive. METHODS: Dictyostelium cells with different genetic backgrounds were treated with direct current electric signals to investigate the genetic regulation of electrotaxis. RESULTS: We demonstrated that electric signals promoted PTEN phosphatase activity and asymmetrical translocation to the posterior plasma membrane of the electrotaxing cells. Electric stimulation produced a similar but delayed rear redistribution of myosin II, immediately before electrotaxis started. Actin polymerization is required for the asymmetric membrane translocation of PTEN and myosin. PTEN signaling is also responsible for the asymmetric anterior redistribution of PIP3/F-actin, and a biased redistribution of pseudopod protrusion in the forwarding direction of electrotaxing cells. CONCLUSIONS: PTEN controls electrotaxis by coordinately regulating asymmetric redistribution of myosin to the posterior, and PIP3/F-actin to the anterior region of the directed migration cells.

Alternative radiopacifiers for polymethyl methacrylate bone cements: Silane-treated anatase titanium dioxide and yttria-stabilised zirconium dioxide.

Poly (methyl methacrylate) (PMMA) bone cement is widely used for anchoring joint arthroplasties. In cement brands approved for these procedures, micron-sized particles (usually barium sulphate, BaSO4) act as the radiopacifier. It has been postulated that these particles act as sites for crack initiation and subsequently cement fatigue. This study investigated whether alternative radiopacifiers, anatase titanium dioxide (TiO2) and yttria-stabilised zirconium dioxide (ZrO2), could improve the in vitro mechanical, fatigue crack propagation and biological properties of polymethyl methacrylate (PMMA) bone cement and whether their coating with a silane could further enhance cement performance. Cement samples containing 0, 5, 10, 15, 20 and 25%w/w TiO2 or ZrO2 and 10%w/w silane-treated TiO2 or ZrO2 were prepared and characterised in vitro in terms of radiopacity, compressive and bending strength, bending modulus, fatigue crack propagation, hydroxyapatite forming ability and MC3T3-E1 cell attachment and viability. Cement samples with greater than 10%w/w TiO2 and ZrO2 had a similar radiopacity to the control 10%w/w BaSO4 cement and commercial products. The addition of TiO2 and ZrO2 to bone cement reduced the bending strength and fracture toughness and increased fatigue crack propagation due to the formation of agglomerations and voids. Silane treating TiO2 reversed this effect, enhancing the dispersion and adhesion of particles to the PMMA matrix and resulted in improved mechanical properties and fatigue crack propagation resistance. Silane-treated TiO2 cements had increased nucleation of hydroxyapatite and MC3T3-E1 cell attachment in vitro, without significantly compromising cell viability. This research has demonstrated that 10%w/w silane-treated anatase TiO2 is a promising alternative radiopacifier for PMMA bone cement offering additional benefits over conventional BaSO4 radiopacifiers.

Liposomes loaded with transforming growth factor ß1 promote odontogenic differentiation of dental pulp stem cells.

OBJECTIVES: This study investigated whether novel liposome formulations loaded with transforming growth factor ß1 (TGF-ß1) could promote the odontogenic differentiation of human dental pulp stem cells (hDPSCs) for dentine-pulp regeneration. METHODS: 0-100 ng/mL of liposomal TGF-ß1 was prepared using the thin-film hydration method. Release of TGF-ß1 from the liposomes was quantified by an enzyme-linked immunosorbent assay (ELISA). The hDPSCs were treated with different concentrations of liposomal TGF-ß1 and cell viability was tested using an MTT assay. "Osteodentine" differentiation capacity was assessed by RT-qPCR, ELISA and Alizarin red S staining. RESULTS: The ELISA results showed that liposomal TGF-ß1 achieved a controlled and prolonged release over time. The MTT results demonstrated that the liposomes (100 µg/mL) were not cytotoxic to the cells. Liposomal TGF-ß1 up-regulated the expression of "osteodentine" markers, RUNX-2, DMP-1 and DSPP, in hDPSCs after 7 days of treatment and resulted in the accumulation of mineralised nodules. CONCLUSION: This study indicated that liposomes are an effective carrier for delivering TGF-ß1 over time. Liposomal TGF-ß1 promoted dentinogenesis and increased mineralisation in hDPSCs. This highlights the potential of liposomal TGF-ß1 for future use in dentine-pulp regeneration. CLINICAL SIGNIFICANCE: Liposomal TGF-ß1 may be used as a synergist for promoting dentine-pulp regeneration of immature permanent teeth or as a pulp capping agent for inducing reparative dentine formation.

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.

Initial Investigations of the Cranial Size and Shape of Adult Eurasian Otters (Lutra lutra) in Great Britain.

Three-dimensional (3D) surface scans were carried out in order to determine the shapes of the upper sections of (skeletal) crania of adult Eurasian otters (Lutra lutra) from Great Britain. Landmark points were placed on these shapes using a graphical user interface (GUI) and distance measurements (i.e., the length, height, and width of the crania) were found by using the landmark points. Male otters had significantly larger skulls than females (P < 0.001). Differences in size also occurred by geographical area in Great Britain (P < 0.05). Multilevel Principal Components Analysis (mPCA) indicated that sex and geographical area explained 31.1% and 9.6% of shape variation in "unscaled" shape data and that they explained 17.2% and 9.7% of variation in "scaled" data. The first mode of variation at level 1 (sex) correctly reflected size changes between males and females for "unscaled" shape data. Modes at level 2 (geographical area) also showed possible changes in size and shape. Clustering by sex and geographical area was observed in standardized component scores. Such clustering in a cranial shape by geographical area might reflect genetic differences in otter populations in Great Britain, although other potentially confounding factors (e.g., population age-structure, diet, etc.) might also drive regional differences. This work provides a successful first test of the effectiveness of 3D surface scans and multivariate methods, such as mPCA, to study the cranial morphology of otters.

Liposomal Delivery of Demineralized Dentin Matrix for Dental Tissue Regeneration.

Current dental restorations have short longevity, and consequently, there is a need for novel tissue engineering strategies that aim to regenerate the dentin-pulp complex. Dentin matrix contains a myriad of bioactive growth factors and extracellular matrix proteins associated with the recruitment, proliferation, and differentiation of dental pulp progenitor cells. In this study, we show that demineralized dentin matrix (DDM), from noncarious dentine, can be encapsulated into liposomes for delivery to dental tissue to promote regeneration. Liposomes were formulated to encapsulate 0-100 µg/mL DDM, lysed with Triton X, and used in vascular endothelial growth factor (VEGF) and transforming growth factor-ß1 (TGF-ß1) enzyme-linked immunosorbent assays to quantify release. The encapsulation efficiencies were calculated to be 25.9% and 28.8% (VEGF/TGF-ß1) for 50 µg/mL DDM liposomes and 39% and 146.7% (VEGF/TGF-ß1) for 100 µg/mL DDM liposomes. All liposome formulations had no cytotoxic effects on a dental pulp stem cell (DPSC) clone, as shown by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide), Caspase 3/7 assays, and cell counts. The ability of the liposomes to stimulate DPSC chemotactic recruitment was tested by Boyden chamber chemotaxis assays. Unloaded liposomes alone stimulated significant progenitor cell recruitment, while DDM-loaded liposomes further promoted chemotactic recruitment in a dose-dependent manner. DDM liposomes promoted the upregulation of "osteodentin" markers osteocalcin and RUNX2 (Runt-related transcription factor 2) in DPSCs after 9 days of treatment, determined by real-time quantitative PCR. Furthermore, Alizarin Red S staining showed that unloaded liposomes alone induced biomineralization of DPSCs, and DDM liposomes further increased the amount of mineralization observed. DDM liposomes were more effective than free DDM (10 µg/mL) at activating recruitment and osteogenic differentiation of DPSC, which are key events in the endogenous repair of the dentin-pulp complex. The study has highlighted the therapeutic potential of bioactive DDM liposomes in activating dental tissue repair in vitro, suggesting that liposomal delivery from biomaterials could be a valuable tool for reparative dentistry and hard-tissue engineering applications.

An assessment of early colonisation of implant-abutment metal surfaces by single species and co-cultured bacterial periodontal pathogens.

OBJECTIVE: Numerous studies have proposed that smooth metal surfaces reduce initial bacterial attachment in the establishment of an early biofilm formation. However, these studies have largely examined single bacterial species, which are not always relevant as pathogens identified as initiators of inflammatory peri-implantitis. This study investigated the adherence of four periodontally-relevant bacterial species to implant and abutment surfaces in current clinical use. METHODS: Discs of polished cobalt chromium (CoCr-polished) and milled titanium (Ti-milled), representing two clinically relevant surfaces, were prepared and surfaces were characterised. Bacterial species Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia and Aggregatibacter actinomycetemcomitans were cultured to mid-log or stationary growth phase. Co-cultures of P. gingivalis, F. nucleatum and P. gingivalis, F. nucleatum, Pr. intermedia were similarly prepared. Bacteria were inoculated onto discs for 2h, stained with a live/dead fluorescent stain and percentage bacterial coverage was calculated by confocal microscopy and image analysis. RESULTS: CoCr-polished discs had smooth surfaces with gentle valley structures, whilst Ti-milled discs had sharp edged peaks. Both discs demonstrated a partial wetting ability capable of initiating bacterial adhesion. P. gingivalis, F. nucleatum and co-cultures, at both mid-log and stationary concentrations, demonstrated equally high coverage of both the smooth CoCr-polished and the rougher Ti-milled metal surfaces. Pr. intermedia and A. actinomycetemcomitans demonstrated lower surface coverage which was slightly higher for Ti-milled. CONCLUSION: Variability was noted in the adherence potential for the respective periodontal pathogens examined. Particularly high adherence was noted for P. gingivalis and F. nucleatum, despite the manufacture of a smooth surface. CLINICAL SIGNIFICANCE: Both surfaces studied may be used at implant-abutment junctions and both possess an ability to establish a bacterial biofilm containing a periodontally-relevant species. These surfaces are thus able to facilitate the apical migration of bacteria associated with peri-implantitis.

A novel liposomal drug delivery system for PMMA bone cements.

The population in developed countries is ageing and the number of people experiencing joint-related conditions, such as osteoarthritis, is expected to increase. Joint replacements are currently the most effective treatment for severe joint conditions and although many of these procedures are successful, infection developing after the procedure is still an issue, requiring complex and expensive revisions. Whilst incorporating a powdered antibiotic within the bone cement can reduce infection rates, the powder frequently agglomerates, resulting in poor antibiotic release characteristics and compromised mechanical performance of the cement. To overcome these issues, a novel delivery system consisting of antibiotic-loaded nano-sized liposomes was developed for inclusion into polymethyl methacrylate (PMMA) bone cement. This system was tested in a commercial cement (Palacos R) and consistently delivered a higher percentage (22%) of the incorporated antibiotic when compared to the powdered antibiotic cement (9%), meaning less antibiotic needs to be incorporated than with conventional cement. The novel system resulted in a controlled and gradual release of antibiotic over a longer, 30-day period and enhanced the toughness, bending strength and Vickers hardness of the cement, without altering its polymerization or molecular structure. This new material has the potential to significantly reduce infections in cemented joint replacements leading to enhanced patient quality of life and reduced healthcare costs. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1510-1524, 2016.

Fluorophosphonate-functionalised titanium via a pre-adsorbed alkane phosphonic acid: a novel dual action surface finish for bone regenerative applications.

Enhancing vitamin D-induced human osteoblast (hOB) maturation at bone biomaterial surfaces is likely to improve prosthesis integration with resultant reductions in the need for revision arthroplasty consequent to aseptic loosening. Biomaterials that are less appealing to microorganisms implicated in implant failures through infection are also highly desirable. However, finding surfaces that enhance hOB maturation to active vitamin D yet deter bacteria remain elusive. In addressing this, we have sought to bio-functionalise titanium (Ti) with lysophosphatidic acid (LPA) and related, phosphatase-resistant, LPA analogues. The impetus for this follows our discovery that LPA co-operates with active vitamin D3 metabolites to secure hOB maturation in vitro including cells grown upon Ti. LPA has also been found, by others, to inhibit virulence factor production and biofilm formation of the human opportunistic pathogen Pseudomonas aeruginosa. Collectively, selected LPA species might offer potential dual-action surface finishes for contemporary bone biomaterials. In attaching a phosphatase-resistant LPA analogue to Ti we took advantage of the affinity of alkane phosphonic acids for TiO2. Herein, we provide evidence for the facile development of a dual-action Ti surface for potential orthopaedic and dental applications. Successful conjugation of an LPA analogue (3S)1-fluoro-3-hydroxy-4-(oleoyloxy)butyl-1-phosphonate (FHBP) to the Ti surface was supported through physiochemical characterisation using x-ray photoelectron spectroscopy and secondary ion mass spectrometry. hOB maturation to active vitamin D3 was enhanced for cells grown on FHBP-Ti whilst these same surfaces exhibited clear antiadherent properties towards a clinical isolate of Staphylococcus aureus.

Elucidating the cellular actions of demineralised dentine matrix extract on a clonal dental pulp stem cell population in orchestrating dental tissue repair.

Bioactive growth factors identified within the extracellular matrix of dentine have been proposed roles in regulating the naturally inherent regenerative dentine formation seen in teeth in response to trauma and infection, which may also be harnessed for novel clinical treatments in augmenting mineralised tissue repair. This study examined the specific biological action of demineralised dentine matrix extract on a clonal population of dental pulp stem cells in stimulating the prerequisite stages of wound healing associated with mineralised tissue repair. A clonal dental pulp stem cell population with sustained proliferative capacity and multi-potentiality towards osteogenic, adipogenic and chondrogenic lineages was isolated from the pulp of human third molars. Dentine was collected from human healthy teeth, powdered and treated with ethylenediaminetetraacetic acid to obtain a solubilised DDM protein extract. The influence of DDM on the DPSC clonal population was assessed in vitro. Exposure of cells to proteolytically degraded DDM or unsupplemented media served as controls. Compared to controls, DDM stimulated cell expansion, reduced apoptotic marker caspase 3, increased cell survival marker Akt1 and enhanced mineralised matrix deposition as determined by mineral deposition and increased expression of bone-related markers, alkaline phosphatase and osteopontin. Dental pulp stem cells successfully migrated into collagen gels supplemented with demineralised dentine matrix, with cells remaining viable and expanding in numbers over a 3-day period. Collectively, the results provide evidence that soluble proteins extracted from dentine matrix are able to exert a direct biological effect on dental pulp stem cells in promoting mineralised tissue repair mechanisms.

Ageing and moisture uptake in polymethyl methacrylate (PMMA) bone cements.

Bone cements are extensively employed in orthopaedics for joint arthroplasty, however implant failure in the form of aseptic loosening is known to occur after long-term use. The exact mechanism causing this is not well understood, however it is thought to arise from a combination of fatigue and chemical degradation resulting from the hostile in vivo environment. In this study, two commercial bone cements were aged in an isotonic fluid at physiological temperatures and changes in moisture uptake, microstructure and mechanical and fatigue properties were studied. Initial penetration of water into the cement followed Fickian diffusion and was thought to be caused by vacancies created by leaching monomer. An increase in weight of approximately 2% was experienced after 30 days ageing and was accompanied by hydrolysis of poly(methyl methacrylate) (PMMA) in the outermost layers of the cement. This molecular change and the plasticising effect of water resulted in reduced mechanical and fatigue properties over time. Cement ageing is therefore thought to be a key contributor in the long-term failure of cemented joint replacements. The results from this study have highlighted the need to develop cements capable of withstanding long-term degradation and for more accurate test methods, which fully account for physiological ageing.