Cell Homing for Pulp Tissue Engineering with Endogenous Dentin Matrix Proteins.

INTRODUCTION: Compelling evidence pinpoints that pulp tissue engineering after the transplantation of stem cells is possible. Although intriguing, severe problems regarding clinical feasibility remain. Cell homing has been proposed as a viable alternative in which dentin-derived growth factors in a conducive scaffold may attract resident cells to form pulplike tissue. In this study, an ectopic animal model for in situ dental pulp tissue engineering was developed to evaluate whether pulplike tissue formation in empty root canals after the attraction of stem cells was possible and whether this could be enhanced by dentin-derived growth factors. METHODS: Three types of fibrin (custom-made fibrin, fibrin sealant, and plasma rich in growth factors [PRGF]) as well as a self-assembling peptide were evaluated in vivo in a modified tooth root model using human teeth. Root canal dentin was conditioned with EDTA, tooth roots were filled with growth factor-laden scaffolds, and dental pulp stem cells in collagen were placed at the root tip. Constructs were implanted into immunocompromised mice for 4 weeks and subsequently analyzed histologically. Differential interference contrast and second harmonic generation imaging were performed for selected sections. RESULTS: For custom-made fibrin and fibrin sealant with dentin matrix proteins, migration into the roots and the formation of a pulplike tissue were observed, whereas the peptide-based scaffold appeared less suitable. PRGF supported tissue formation regardless of the addition of dentin matrix proteins. In the test groups with dentin matrix proteins and EDTA conditioning, differentiated odontoblastlike cells extended cellular processes into the dentinal tubules, which coincided with the deposition of the newly formed collagenous dentin matrix. CONCLUSIONS: This new cell homing model provides evidence that fibrin derivatives make applicable scaffolds and that dentin-derived proteins induce chemotaxis and pulplike tissue formation.

Suitability of Different Natural and Synthetic Biomaterials for Dental Pulp Tissue Engineering.

Dental pulp tissue engineering is possible after insertion of pulpal stem cells combined with a scaffold into empty root canals. Commonly used biomaterials are collagen or poly(lactic) acid, which are either difficult to modify or to insert into such a narrow space. New hydrogel scaffolds with bioactive, specifically tailored functions could optimize the conditions for this approach. Different synthetic and natural hydrogels were tested for their suitability to engineer dental pulp. Two functionalized modifications of polyethylene glycol were developed in this study and compared to a self-assembling peptide, as well as to collagen and fibrin. Cell viability of dental pulp stem cells in test materials was assessed over two weeks. Cells in selected test materials laden with dentin-derived growth factors were inserted into human tooth roots and implanted subcutaneously into immunocompromised mice. In vitro cell culture exhibited distinct differences between scaffold types, where viability was significantly higher in natural compared to synthetic materials. In vivo experiments showed considerable differences regarding scaffold degradation, soft tissue formation, vascularization, and odontoblast-like cell differentiation. Fibrin appeared most suitable to enable generation of a pulp-like tissue and differentiation of cells into odontoblasts at the cell-dentin interface. In conclusion, natural materials, especially fibrin, proved to be superior compared to synthetic scaffolds regarding cell viability and dental pulp-like tissue formation.

Influence of root canal disinfectants on growth factor release from dentin.

INTRODUCTION: During dentinogenesis, growth factors become entrapped in the dentin matrix that can later be released by demineralization. Their effect on pulpal stem cell migration, proliferation, and differentiation could be beneficial for regenerative endodontic therapies. However, precondition for success, as for conventional root canal treatment, will be sufficient disinfection of the root canal system. Various irrigation solutions and intracanal dressings are available for clinical use. The aim of this study was 2-fold: to identify a demineralizing solution suitable for growth factor release directly from dentin and to evaluate whether commonly used disinfectants for endodontic treatment will compromise this effect. METHODS: Dentin disks were prepared from extracted human teeth and treated with EDTA or citric acid at different concentrations or pH for different exposure periods. The amount of transforming growth factor-ß1 (TGF-ß1), fibroblast growth factor 2, and vascular endothelial growth factor were quantified via enzyme-linked immunosorbent assay and visualized by gold labeling. Subsequently, different irrigation solutions (5.25% sodium hypochloride, 0.12% chlorhexidine digluconate) and intracanal dressings (corticoid-antibiotic paste, calcium hydroxide: water-based and oil-based, triple antibiotic paste, chlorhexidine gel) were tested, and the release of TGF-ß1 was measured after a subsequent conditioning step with EDTA. RESULTS: Conditioning with 10% EDTA at pH 7 rendered the highest amounts of TGF-ß1 among all test solutions. Fibroblast growth factor 2 and vascular endothelial growth factor were detected after EDTA conditioning at minute concentrations. Irrigation with chlorhexidine before EDTA conditioning increased TGF-ß1 release; sodium hypochloride had the opposite effect. All tested intracanal dressings interfered with TGF-ß1 release except water-based calcium hydroxide. CONCLUSIONS: Growth factors can be released directly from dentin via EDTA conditioning. The use of disinfecting solutions or medicaments can amplify or attenuate this effect.

Probing of microbial biofilm communities for coadhesion partners.

Investigations of interbacterial adhesion in dental plaque development are currently limited by the lack of a convenient assay to screen the multitude of species present in oral biofilms. To overcome this limitation, we developed a solid-phase fluorescence-based screening method to detect and identify coadhesive partner organisms in mixed-species biofilms. The applicability of this method was demonstrated using coaggregating strains of type 2 fimbrial adhesin-bearing actinomyces and receptor polysaccharide (RPS)-bearing streptococci. Specific adhesin/receptor-mediated coadhesion was detected by overlaying bacterial strains immobilized to a nitrocellulose membrane with a suspended, fluorescein-labeled bacterial partner strain. Coadhesion was comparable regardless of which cell type was labeled and which was immobilized. Formaldehyde treatment of bacteria, either in suspension or immobilized on nitrocellulose, abolished actinomyces type 2 fimbrial adhesin but not streptococcal RPS function, thereby providing a simple method for assigning complementary adhesins and glycan receptors to members of a coadhering pair. The method's broader applicability was shown by overlaying colony lifts of dental plaque biofilm cultures with fluorescein-labeled strains of type 2 fimbriated Actinomyces naeslundii or RPS-bearing Streptococcus oralis. Prominent coadhesion partners included not only streptococci and actinomyces, as expected, but also other bacteria not identified in previous coaggregation studies, such as adhesin- or receptor-bearing strains of Neisseria pharyngitis, Rothia dentocariosa, and Kingella oralis. The ability to comprehensively screen complex microbial communities for coadhesion partners of specific microorganisms opens a new approach in studies of dental plaque and other mixed-species biofilms.

Cell-free approaches for dental pulp tissue engineering.

The standard treatment modality for teeth with irreversibly damaged dental pulp is root canal therapy, which involves complete removal of the soft tissue and obturation with a synthetic material. So far, research studies show that the combination of stem cells with a suitable scaffold material and transplantation into the root canal may result in the generation of pulplike tissue and the formation of tubular dentin. Because of the technical challenges associated with such a procedure, cell-free alternatives that take advantage of the dental pulp's inherent regenerative capacity because of endogenous stem cell populations and bioactive dentin matrix components need to be considered and explored. Following the tissue engineering approach, this includes (1) a bioactive scaffold, (2) growth and differentiation factors from dentin, and (3) the recruitment of stem cells from resident populations within the pulp or from the periapical region. If this concept proved to be successful, cell-free therapies may be a safer, more practical, feasible, and affordable approach to dental pulp regeneration.

Salivary protein adsorption and Streptococccus gordonii adhesion to dental material surfaces.

OBJECTIVES: The initial adhesion of microorganisms to clinically used dental biomaterials is influenced by physico-chemical parameters like hydrophobicity and pre-adsorption of salivary proteins. Here, polymethyl methacrylate (PMMA), polyethylene (PE), polytetrafluoroethylene (PTFE), silicone (Mucopren soft), silorane-based (Filtek Silorane) and methacrylate-based (Tetric EvoCeram) dental composites, a conventional glassionomer cement as well as cobalt-chromium-molybdenum (Co28Cr6Mo) and titanium (Ti6Al4V) were tested for adsorption of salivary proteins and adhesion of Streptococcus gordonii DL1. METHODS: Wettability of material surfaces precoated with salivary proteins or left in phosphate-buffered saline was determined by the measurement of water contact angles. Amounts of adsorbed proteins were determined directly on material surfaces after biotinylation of amino groups and detection by horseradish peroxidase-conjugated avidin-D. The same technique was used to analyze for the binding of biotinylated bacteria to material surfaces. RESULTS: The highest amount of proteins (0.18µg/cm(2)) adsorbed to hydrophobic PTFE samples, and the lowest amount (0.025µg/cm(2)) was detected on silicone. The highest number of S. gordonii (3.2×10(4)CFU/mm(2)) adhered to the hydrophilic glassionomer cement surface coated with salivary proteins, and the lowest number (4×10(3)CFU/mm(2)) was found on the hydrophobic silorane-based composite. Hydrophobicity of pure material surfaces and the number of attached microorganisms were weakly negatively correlated. No such correlation between hydrophobicity and the number of bacteria was detected when surfaces were coated with salivary proteins. SIGNIFICANCE: Functional groups added by the adsorption of specific salivary proteins to material surfaces are more relevant for initial bacterial adhesion than hydrophobicity as a physical property.

Influences of protein films on antibacterial or bacteria-repellent surface coatings in a model system using silicon wafers.

Immobilization of defined chemical functionalities to biomaterial surfaces is employed to optimize them not only for tissue compatibility but also for prevention of bacterial infection. Grafting surfaces with chains of poly(ethylene glycol) (PEG) results in bacterial repellence whereas modification with cationic groups conveys them with bactericidal properties. Since biomaterials in situ will become exposed to a protein-rich environment, it is necessary to investigate the influence of prior protein adsorption on the antibacterial activity of this type of chemical surface modification. In the present study, we immobilized short-chain PEG and two pyridinium group-containing methacrylate monomers, 12-methacryloyloxydodecylpyridinium bromide (MDPB) and 6-methacryloyloxyhexylpyridinium chloride (MHPC), to silicon wafer model surfaces to investigate the influence of prior protein adsorption on the bactericidal activity of the surface coating towards subsequently attached bacteria. Adsorbed amounts of human serum albumin and salivary proteins were found to be two times higher on cationic compared to PEG-modified surfaces. An analogous tendency was found for attachment of Streptococcus gordonii and Streptococcus mutans to the same surfaces without prior protein exposure. However, most bacteria attached to cationic surfaces were found to be dead. Prior exposure of cationic surfaces to protein solutions drastically altered bacterial attachment dependent on the type of protein solution and bacterial species employed. Significantly, the original bactericidal activity of pyridinium-coated surfaces was found greatly reduced upon adsorption of a protein film. As a conclusion we propose that future approaches should combine the protein- and bacteria-repellent properties of PEG-coatings with the bactericidal function of charged cationic groups.

The anti-adherence activity and bactericidal effect of microparticulate silver additives in composite resin materials.

OBJECTIVE: Resin composite materials tend to accumulate microorganisms and dental plaque, which in turn may induce secondary caries around adhesive restorations. The aim of the present in vitro study was to evaluate the antibacterial activity of a resin composite material loaded with silver microparticles against Streptococcus mutans. DESIGN: Circular specimens (10.0mm in diameter) of a resin composite matrix loaded with two different concentrations of a silver additive (Comp0.3: 0.3%; Comp0.6: 0.6%) and one unloaded reference composite matrix (Comp0: 0%) were made. Surface roughness R(a) was assessed by perthometer measurements and hydrophobicity according to water contact angles was determined by computerized image analysis. The specimens were incubated in a S. mutans suspension (1h, 37 degrees C) and adhering streptococci were quantified by using a biofluorescence assay (Alamar blue/Resazurin). Additionally, the viability of adhering bacteria was assessed by live/dead cell labelling in combination with fluorescence microscopy. RESULTS: Statistically significant differences between the median water contact angles of Comp0 (66.3 degrees ), Comp0.3 (76.7 degrees ), and Comp0.6 (89.4 degrees ) were observed (p<0.001). A three- to fourfold higher amount of adhering S. mutans was found on reference Comp0 (12,093relative fluorescence units) than on Comp0.3 (4258rfu) and Comp0.6 (3292) (p<0.001 for both). Significantly higher percentages of dead cells than on Comp0 (0.5%) were found on Comp0.3 (6.1%) and on Comp0.6 (10.1%) (p<0.001 for both). CONCLUSIONS: The addition of microparticulate silver to a resin composite material increased the surface hydrophobicity and reduced the number of adhering streptococci. Simultaneously it increased the percentage of dead and inactive cells on the composite surface. Thus, silver additives seem to demonstrate anti-adherence activity as well as a bactericidal effect.