Dental Tissue Engineering Applications Research Articles

Electrospun bioactive tertiary glass nanoparticles-containing silica/gelatin/polyethylene oxide hybrid membranes for potential dental bone tissue engineering applications

Silica (SiO2)/gelatin (G)/polyethylene oxide (PEO) hybrid membranes with maximized gelatin and bioactive glass nanoparticles (NpBG) contents were produced by electrospinning using water as main solvent. The membranes obtained from the precursor 80 %SiO2/10 %G/8%PEO/2%NpBG were bioactive, non-toxic and showed enhanced ability for osteogenic differentiation, demonstrating their potential for dental tissue engineering applications.

Role of magnesium on phase composition of tricalcium phosphate and its interaction with human dental pulp stem cells

Tricalcium phosphates (TCPs) are of great interest in dental tissue engineering applications. The objective of this study was to incorporate magnesium (Mg) at different concentrations of 0.25, 0.50, 1.00, 2.50, and 5.00 wt.% to TCP and evaluate the effects on phase composition, density, and in vitro interaction with human dental pulp stem cells (hDPSCs). Our results showed that adding Mg stabilized the β-TCP crystal structure and increased the density of the TCP. Mg addition had no adverse effect on hPDSCs response. Although cellular proliferation was slightly less on Mg-TCPs at early time points, it increased significantly with time and in a dose-dependent manner. A similar trend was observed for cellular differentiation. Together, these data show for the first time that Mg addition at concentrations less than 2.50% (preferably at 1.00 or 2.50 wt.%) can be added safely to TCP for enhanced proliferation and differentiation of hDPSCs.Graphical abstract

Role of iron on physical and mechanical properties of brushite cements, and interaction with human dental pulp stem cells

Improving the physical, mechanical and biological properties of brushite cements (BrC) is of a great interest for using them in bone and dental tissue engineering applications. The objective of this study was to incorporate iron (Fe) at different concentrations (0.25, 0.50, and 1.00 wt%) to BrC and study the role of Fe on phase composition, setting time, compressive strength, and interaction with human dental pulp stem cells (hDPSCs). Results showed that increase in Fe concentration increases the β-tricalcium phosphate (β-TCP)/dicalcium phosphate dihydrate (DCPD) ratio and prolongs the initial and final setting time due to effective role of Fe on stabilizing the β-TCP crystal structure and retarding its dissolution kinetic, in a dose dependent manner where the highest setting time was recorded for 1.00 wt% Fe–BrC sample. Addition of low concentrations of Fe (0.25 and 0.50 wt%) did not have adverse effect on compressive strength and strength was in the range of 5.7–7.05 (±~1.4) MPa; however, presence of 1.00 wt% Fe decreases the strength of BrC from 7.05 ± 1.57 MPa to 3.12 ± 1.06 MPa. Interaction between the BrCs and hDPSCs was evaluated by cell proliferation assay, scanning electron microscopy, and live/dead staining. Low concentrations of 0.25, and 0.50 wt% of Fe did not have any adverse effect on cell attachment and proliferation; while significant decrease in cellular activity was evident in BrC samples doped with 1.00 wt %. Together, these data show that low concentrations of Fe (equal or less than 0.50 wt %) can be safely added to BrC without any adverse effect on physical, mechanical and biological properties in presence of hDPSCs.

The Applications of 3D Printing for Craniofacial Tissue Engineering.

Three-dimensional (3D) printing is an emerging technology in the field of dentistry. It uses a layer-by-layer manufacturing technique to create scaffolds that can be used for dental tissue engineering applications. While several 3D printing methodologies exist, such as selective laser sintering or fused deposition modeling, this paper will review the applications of 3D printing for craniofacial tissue engineering; in particular for the periodontal complex, dental pulp, alveolar bone, and cartilage. For the periodontal complex, a 3D printed scaffold was attempted to treat a periodontal defect; for dental pulp, hydrogels were created that can support an odontoblastic cell line; for bone and cartilage, a polycaprolactone scaffold with microspheres induced the formation of multiphase fibrocartilaginous tissues. While the current research highlights the development and potential of 3D printing, more research is required to fully understand this technology and for its incorporation into the dental field.

Evaluation of human dental pulp stem cells behavior on a novel nanobiocomposite scaffold prepared for regenerative endodontics

Dental caries is a dental disease affecting public health, which results in many socio-economic consequences. This disease causes loss of tooth hard tissue and subsequent inflammation and loss of the dental pulp. In this study, it was aimed to develop and characterize boron (B) modified bioactive glass nanoparticles (BG-NPs) containing cellulose acetate/oxidized pullulan/gelatin (CA/ox-PULL/GEL) three dimensional scaffolds with tubular morphology for dentin regeneration. 3D nanobiocomposite structures were prepared by thermally induced phase separation and porogen leaching methods and characterized by in vitro degradation analysis, water absorption (WA) capacity measurement, SEM, in vitro biomineralization analysis, porosity measurement and mechanical tests. Scaffolds lost about (30–40)% of their weight during one month and WA capacity decreased with increase in immersion time in phosphate buffer solution (PBS) during one month. According to SEM, aligned and tubular structures were formed with mean diameter of about 11 μm, and BG-NPS were distributed evenly in all parts of the scaffolds. Scaffolds (without BG-NPs) possessed the highest porosity percentage. Addition of 10% BG-NPs improved the mechanical properties of scaffolds. Scaffolds surfaces were fully covered by calcium phosphate (Ca-P) deposits after conditioning in simulated body fluid for 14 days with higher quantity of deposition in groups with inclusion of B-BG-NPs. Human dental pulp stem cells (hDPSCs) were isolated from third molar teeth and used in cell culture studies. In all groups, cells adhered well 1 day after culture. Group B14–10 showed a slight increase of proliferation than group (without BG-NPs) after 7 days of incubation. Alkaline phosphatase activity (ALP) and intracellular calcium amounts increased significantly 14 days after incubation with highest values in B14–10 and B14–20 groups. Confocal laser scanning microscopy (CSLM) analysis, showed that cells on B14–10 and B21–10 scaffold groups, spread more 14 days after culture, and they also possessed extended processes specific to odontoblasts. Alizarin Red quantification showed that the highest calcium deposition was observed on B14–10 scaffolds. Immunohistochemical and Von Kossa stainings showed that scaffolds positively affected the odontoblastic differentiation of the hDPSCs. In this work, results showed that boron modified BG-NPs (B-BG-NPs) incorporated dentin-like constructs bring a new approach for dental tissue engineering applications.

In Vitro Osteogenic, Angiogenic, and Inflammatory Effects of Copper in β-Tricalcium Phosphate

AbstractTricalcium phosphate (TCP) is a promising candidate in bone and dental tissue engineering applications. Though osteoconductive, its low osteoinductivity is a major concern. Trace elements addition at low concentrations are known for their impact on not only the osteoinductivity, but also physical and mechanical properties of TCP. Copper (Cu) is known for its role in vascularization and angiogenesis in biological systems. Here, we studied the effects of Cu addition on phase composition, porosity, microstructure and in vitro interaction with osteoblast (OB) cells. Our results showed that Cu stabilized the TCP structure, while no significant effect of microstructure and porosity was found. Cu at concentrations less than 1 wt.% did not have any cytotoxic effect while decreased proliferation of OBs were observed at 1 wt.% Cu doped TCP. Addition of Cu upregulated collagen type I and vascular endothelial growth factor expression in a dose dependent manner at early time-point. Furthermore, Cu reduced inflammatory gene expression by human osteoblasts. These findings show that addition of Cu to TCP may provide a therapeutic strategy that can be applied in bone tissue engineering applications.

Antibacterial Nanostructured Polyhydroxybutyrate Membranes for Guided Bone Regeneration.

The principle of guided bone regeneration (GBR) in orthopedic, cranio-maxillofacial and dental tissue engineering applications is to create a secluded space for the treatment of large bone defects while excluding fibrous connective tissue formation at the defect area. In dental surgeries, a GBR membrane is placed near the dental implant in post-extraction sockets to grow new bone at the implant site, along with inhibiting infection due to the microbial nature of the mouth flora. Poly[(R)-3-hydroxybutyric acid] (PHB) is a natural polyester synthesized by a wide variety of microorganisms which has been proposed for various biomedical applications. In this study, to improve the performance of PHB as a GBR, a NaOH based alkaline treatment was designed to create nanofeatured PHB membranes. The newly fabricated nanofeatured PHB membranes were investigated for GBR applications. The results showed that a quick, simple, and inexpensive sodium hydroxide treatment modified the nanostructured surface morphology and chemistry of the PHB membranes by inducing hydrolysis of the ester bonds in the PHB backbone creating carboxylic surface functional groups, which increased the hydrophilicity of the PHB surfaces. Cytocompatibility studies showed increased proliferation of human osteoblasts (bone forming cells) on the NaOH treated PHB membranes compared to the untreated ones. Importantly, in vitro bacterial studies with Staphylococcus aureus (S. aureus) indicated that the NaOH-treated PHB surfaces inhibited S. aureus growth more than 60% after 48 hours of culture compared to the untreated PHB membrane. Thus, this study, for the first time, showed that nanofeatured PHB membranes modified with a NaOH treatment may be a useful anti-bacterial, osteoconductive GBR membrane for numerous orthopedic, cranio-maxillofacial and dental tissue engineering applications.

PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells.

The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

Polyetheretherketone/nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties

Lack of antibacterial activity and binding ability to natural bone tissue has significantly limited polyetheretherketone (PEEK) for many challenging dental implant applications. Here, we have developed a polyetheretherketone/nano-fluorohydroxyapatite (PEEK/nano-FHA) biocomposite with enhanced antibacterial activity and osseointegration through blending method. Smooth and rough surfaces of PEEK/nano-FHA biocomposites were also prepared. Our results showed that in vitro initial cell adhesion and proliferation on the nano-FHA reinforced PEEK composite were improved. In addition, higher alkaline phosphatase activity and cell mineralization were also detected in cells cultured on PEEK/nano-FHA biocomposites, especially for rough PEEK/nano-FHA surfaces. More importantly, the as-prepared PEEK/nano-FHA biocomposite could effectively prevent the proliferation and biofilm formation of bacterial. For in vivo test, the newly formed bone volume of PEEK/nano-FHA group was higher than that of bare PEEK group based on 3D microcomputed tomography and 2D histomorphometric analysis. These reports demonstrate that the developed PEEK/nano-FHA biocomposite has increased biocompatibility and antibacterial activity in vitro, and promoted osseointegration in vivo, which suggests that it holds potential to be applied as dental implant material in dental tissue engineering applications.

Characterization of natural, decellularized and reseeded porcine tooth bud matrices

Dental tissue engineering efforts have yet to identify scaffolds that instruct the formation of bioengineered teeth of predetermined size and shape. Here we investigated whether extracellular matrix (ECM) molecules present in natural tooth scaffolds can provide insight on how to achieve this goal. We describe methods to effectively decellularize and demineralize porcine molar tooth buds, while preserving natural ECM protein gradients. Natural tooth ECM composition was assessed using histological and immunohistochemical (IHC) analyses of fibrillar and basement membrane proteins. Our results showed that Collagen I, Fibronectin, Collagen IV, and Laminin gradients were detected in natural tooth tissues, and retained in decellularized samples. Second harmonic generation (SHG) image analysis and 3D reconstructions were used to show that natural tooth tissue exhibited higher collagen fiber density, and less oriented and less organized collagen fibers, as compared to decellularized tooth tissue. We also found that reseeded decellularized tooth scaffolds exhibited distinctive collagen content and organization as compared to decelluarized scaffolds. Our results show that SHG allows for quantitative assessment of ECM features that are not easily characterized using traditional histological analyses. In summary, our results demonstrate the potential for natural decellularized molar tooth ECM to instruct dental cell matrix synthesis, and lay the foundation for future use of biomimetic scaffolds for dental tissue engineering applications.

Tooth Tissue Engineering: Optimal Dental Stem Cell Harvest Based on Tooth Development

Our long-term objective is to devise reliable methods to generate biological replacement teeth exhibiting the physical properties and functions of naturally formed human teeth. Previously, we demonstrated the successful use of tissue engineering approaches to generate small, bioengineered tooth crowns from harvested pig and rat postnatal dental stem cells (DSCs). To facilitate characterizations of human DSCs, we have developed a novel radiographic staging system to accurately correlate human third molar tooth developmental stage with anticipated harvested DSC yield. Our results demonstrated that DSC yields were higher in less developed teeth (Stages 1 and 2), and lower in more developed teeth (Stages 3, 4, and 5). The greatest cell yields and colony-forming units (CFUs) capability was obtained from Stages 1 and 2 tooth dental pulp. We conclude that radiographic developmental staging can be used to accurately assess the utility of harvested human teeth for future dental tissue engineering applications.