Kerstin M. Galler

Self-assembling multidomain peptide hydrogels: Designed susceptibility to enzymatic cleavage allows enhanced cell migration and spreading

Multidomain peptides are a class of amphiphilic self-assembling peptides with a modular ABA block motif in which the amphiphilic B block drives self-assembly while the flanking A blocks, which are electrostatically charged, control the conditions under which assembly takes place. Previously we have shown that careful selection of the amino acids in the A and B blocks allow one to control the self-assembled fiber length and viscoelastic properties of formed hydrogels. Here we demonstrate how the modular nature of this peptide assembler can be designed for biological applications. With control over fiber length and diameter, gelation conditions, and viscoelastic properties, we can develop suitable materials for biological applications. Going beyond a simple carrier for cell delivery, a biofunctional scaffold will interact with the cells it carries, promoting advantageous cell-matrix interactions. We demonstrate the design of a multidomain peptide into a bioactive variant by incorporation of a matrix metalloprotease 2 (MMP-2) specific cleavage site and cell adhesion motif. Gel formation and rheological properties were assessed and compared to related peptide hydrogels. Proteolytic degradation by collagenase IV was observed in a gel weight loss study and confirmed by specific MMP-2 degradation monitored by mass spectrometry and cryo-transmission electron microscopy (cryo-TEM). Combination of this cleavage site with the cell adhesion motif RGD resulted in increased cell viability and cell spreading and encouraged cell migration into the hydrogel matrix. Collectively the structural, mechanical, and bioactive properties of this multidomain peptide hydrogel make it suitable as an injectable material for a variety of tissue engineering applications.

Self-Assembling Peptide Amphiphile Nanofibers as a Scaffold for Dental Stem Cells

Dental caries remains one of the most prevalent infectious diseases in the world. So far, available treatment methods rely on the replacement of decayed soft and mineralized tissue with inert biomaterials alone. As an approach to develop novel regenerative strategies and engineer dental tissues, two dental stem cell lines were combined with peptide-amphiphile (PA) hydrogel scaffolds. PAs self-assemble into three-dimensional networks of nanofibers, and living cells can be encapsulated. Cell-matrix interactions were tailored by incorporation of the cell adhesion sequence RGD and an enzyme-cleavable site. SHED (stem cells from human exfoliated deciduous teeth) and DPSC (dental pulp stem cells) were cultured in PA hydrogels for 4 weeks using different osteogenic supplements. Both cell lines proliferate and differentiate within the hydrogels. Histologic analysis shows degradation of the gels and extracellular matrix production. However, distinct differences between the two cell lines can be observed. SHED show a spindle-shaped morphology, high proliferation rates, and collagen production, resulting in soft tissue formation. In contrast, DPSC reduce proliferation, but exhibit an osteoblast-like phenotype, express osteoblast marker genes, and deposit mineral. Since the hydrogels are easy to handle and can be introduced into small defects, this novel system might be suitable for engineering both soft and mineralized matrices for dental tissue regeneration.

Somatic stem cells for regenerative dentistry

Complex human tissues harbour stem cells and/or precursor cells, which are responsible for tissue development or repair. Recently, dental tissues such as periodontal ligament (PDL), dental papilla or dental follicle have been identified as easily accessible sources of undifferentiated cells. The dental stem cell biology might provide meaningful insights into the development of dental tissues and cellular differentiation processes. Dental stem cells could also be feasible tools for dental tissue engineering. Constructing complex structures like a periodontium, which provides the functional connection between a tooth or an implant and the surrounding jaw, could effectively improve modern dentistry. Dental precursor cells are attractive for novel approaches to treat diseases like periodontitis, dental caries or to improve dental pulp healing and the regeneration of craniofacial bone and teeth. These cells are easily accessible and, in contrast to bone-marrow-derived mesenchymal stem cells, are more closely related to dental tissues. This review gives a short overview of stem cells of dental origin.

Scaffolds for Dental Pulp Tissue Engineering

For tissue engineering strategies, the choice of an appropriate scaffold is the first and certainly a crucial step. A vast variety of biomaterials is available: natural or synthetic polymers, extracellular matrix, self-assembling systems, hydrogels, or bioceramics. Each material offers a unique chemistry, composition and structure, degradation profile, and possibility for modification. The role of the scaffold has changed from passive carrier toward a bioactive matrix, which can induce a desired cellular behavior. Tailor-made materials for specific applications can be created. Recent approaches to generate dental pulp rely on established materials, such as collagen, polyester, chitosan, or hydroxyapatite. Results after transplantation show soft connective tissue formation and newly generated dentin. For dentin-pulp-complex engineering, aspects including vascularization, cell-matrix interactions, growth-factor incorporation, matrix degradation, mineralization, and contamination control should be considered. Self-assembling peptide hydrogels are an example of a smart material that can be modified to create customized matrices. Rational design of the peptide sequence allows for control of material stiffness, induction of mineral nucleation, or introduction of antibacterial activity. Cellular responses can be evoked by the incorporation of cell adhesion motifs, enzyme-cleavable sites, and suitable growth factors. The combination of inductive scaffold materials with stem cells might optimize the approaches for dentin-pulp complex regeneration.

Inhibition of Phosphatidylinositol 3-Kinase Amplifies TEGDMA-induced Apoptosis in Primary Human Pulp Cells

Cytotoxicity of triethylene glycol dimethacrylate (TEGDMA), a co-monomer of dental resinous restorative materials, is firmly established in vitro, but the molecular mechanisms are unknown. Here we examined apoptosis and necrosis induced by TEGDMA in human primary pulp cells. The levels of apoptotic and necrotic cell populations differentially increased after exposure to increasing concentrations of TEGDMA. A two-fold increase in the percentage of apoptotic cells was induced by 1 mmol/L TEGDMA. However, a population shift among cells in apoptosis and necrosis was detected when cell cultures were exposed to 2 mmol/L TEGDMA. Inhibition of the MAP Kinase/ERK pathway had no influence on cell survival, but inhibition of phosphatidylinositol 3 kinase (PI3-Kinase; Akt/protein kinase B) by LY294002 amplified TEGDMA-induced apoptosis. Moreover, Akt phosphorylation was inhibited in the presence of TEGDMA. These results suggest that depression of PI3K signaling may be a primary target in TEGDMA-induced apoptosis.

Bioengineering of dental stem cells in a PEGylated fibrin gel

AIM Postnatal stem cells can generate tooth-specific structures after transplantation in vivo, which makes them a valuable tool for dental tissue engineering. Scaffold materials that are compatible with dental stem cells, injectable and tunable for targeted regeneration are needed. A candidate material is fibrin, a biopolymer critical to hemostasis and wound healing. Rapid degradation of fibrin can be decelerated by modification with polyethylene glycol (PEG), thus creating a hybrid material for cell delivery. The aim of this study was to evaluate the suitability of PEGylated fibrin as a scaffold for dental stem cells. METHODS A PEGylated fibrin hydrogel was combined with stem cells derived from dental pulp or periodontal ligament. Cell proliferation was assessed over a 4-week period, and alkaline phosphatase activity and expression levels of mineralization-associated genes after osteogenic induction were analyzed. Cell morphology, matrix degradation, collagen production and mineral deposition were evaluated by histology. Constructs of PEGylated fibrin with dental pulp stem cells in dentin disks were transplanted in immunocompromised mice for 5 weeks and examined for new tissue formation. RESULTS All cell types proliferated in PEGylated fibrin. After osteogenic induction, alkaline phosphatase activity was higher and osteoblast-specific genes were upregulated. Dentin-specific markers increased in pulp-derived stem cells. Histologic analysis revealed degradation of fibrin, production of a collagenous matrix and mineral deposition. In vivo transplantation rendered a vascularized soft connective tissue similar to dental pulp. CONCLUSION Fibrin allows for the growth and differentiation of dental stem cells, can be inserted into small defects and thus appears to be a promising biomaterial for tissue regeneration in the oral cavity.

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-β1 (TGF-β1), 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-β1 was measured after a subsequent conditioning step with EDTA. RESULTS Conditioning with 10% EDTA at pH 7 rendered the highest amounts of TGF-β1 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-β1 release; sodium hypochloride had the opposite effect. All tested intracanal dressings interfered with TGF-β1 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.

TEGDMA Reduces Mineralization in Dental Pulp Cells

Direct application of dentin bonding agents onto the exposed pulp has been advocated, but in vivo studies indicate a lack of reparative dentin formation. Our objective was to investigate the role of triethylene glycol dimethacrylate (TEGDMA), a commonly used compound in dentin bonding agents, as a potential inhibitor of mineralization. Human pulp cells were exposed to different concentrations of TEGDMA, and expression of the mineralization-related genes collagen I, alkaline phosphatase, bone sialoprotein, osteocalcin, Runx2, and dentin sialophosphoprotein was analyzed. Gene expression studies by real-time polymerase chain-reaction revealed a concentration- and time-dependent decrease of mineralization markers. A subtoxic TEGDMA concentration (0.3 mM) reduced expression levels by 5 to 20% after 4 hrs and by 50% after 12 hrs. Furthermore, alkaline phosphatase activity and calcium deposition were significantly lower in dental pulp cells treated with TEGDMA over 14 days. These findings indicate that even low TEGDMA concentrations might inhibit mineralization induced by dental pulp cells, thus impairing reparative dentin formation after pulp capping with dentin bonding agents.

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 pulps 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.

Establishment of an optimized ex vivo system for artificial root canal infection evaluated by use of sodium hypochlorite and the photodynamic therapy

AIM To establish a refined model of artificially infected root canals and confirm its suitability as a sensitive ex vivo method to assess the efficacy of disinfecting agents. Disinfection was evaluated using sodium hypochlorite (NaOCl), either blocked or unblocked by sodium thiosulphate, and a recently promoted method of disinfection, the antibacterial photodynamic therapy (PDT). METHODOLOGY The roots of bovine incisors were sectioned into three parts, the canals of coronal and middle regions were filled with a suspension of Enterococcus faecalis and the apical region with culture medium. After 7 days, coronal sections were disinfected using NaOCl (0.5%, 1.0% and 3.0% for 30, 60 and 600 s) or a system for photoactivated chemotherapy (PACT; Cumdente, Tübingen, Germany) for antibacterial PDT. Apical sections served as sterile controls and middle sections as bacterial growth controls. In half of the NaOCl-treated specimens, disinfection was arrested. Dentine chips from biopsies at different depths from the central canal towards the periphery were plated and assessed for colony-forming units (CFU). Disinfection was considered biologically relevant if the reduction of CFU was at least three log10 orders of magnitude. RESULTS Colony-forming units of 10³ - 10⁴ in growth controls indicated effective artificial infection. A biologically relevant reduction of CFU was accomplished with unblocked NaOCl, but not after blocking with NaOCl nor with PDT. CONCLUSIONS The system reliably detected disinfection of the root canal and dentinal tubules and proved suitable for ex vivo testing of root canal disinfection. The effect of NaOCl depended on the duration of impact. Under the present experimental conditions, the antibacterial PDT system did not achieve sufficient disinfection.