Effect of Heparan Sulfate on Vasculogenesis and Dentinogenesis of Dental Pulp Stem Cells.

INTRODUCTION: Heparan sulfate (HS) is a major component of dental pulp tissue. We previously reported that inhibiting HS biosynthesis impedes endothelial differentiation of dental pulp stem cells (DPSCs). However, the underlying mechanisms by which exogenous HS induces DPSC differentiation and pulp tissue regeneration remain unknown. This study explores the impact of exogenous HS on vasculogenesis and dentinogenesis of DPSCs both in vitro and in vivo. METHODS: Human-derived DPSCs were cultured in endothelial and odontogenic differentiation media and treated with HS. Endothelial differentiation of DPSCs was investigated by real-time PCR and capillary sprouting assay. Odontogenic differentiation was assessed through real-time PCR and detection of mineralized dentin-like deposition. Additionally, the influence of HS on pulp tissue was assessed with a direct pulp capping model, in which HS was delivered to exposed pulp tissue in rats. Gelatin sponges were loaded with either phosphate-buffered saline or 101-102 µg/mL HS and placed onto the pulp tissue. Following a 28-day period, tissues were investigated by histological analysis and micro-CT imaging. RESULTS: HS treatment markedly increased expression levels of key endothelial and odontogenic genes, enhanced the formation of capillary-like structures, and promoted the deposition of mineralized matrices. Treatment of exposed pulp tissue with HS in the in vivo pulp capping study induced formation of capillaries and reparative dentin. CONCLUSIONS: Exogenous HS effectively promoted vasculogenesis and dentinogenesis of DPSCs in vitro and induced reparative dentin formation in vivo, highlighting its therapeutic potential for pulp capping treatment.

Poly(lactic acid/caprolactone) bilayer membrane achieves bone regeneration through a prolonged barrier function.

Guided bone regeneration (GBR) is a treatment strategy used to recover bone volume. Barrier membranes are a key component of GBR protocols, and their properties can impact treatment outcomes. This study investigated the efficacy of an experimental, slow-degrading, bilayer barrier membrane for application in GBR using in vivo animal models. A synthetic copolymer of poly(lactic acid/caprolactone) (PLCL) was used to prepare a slow-degrading bilayer membrane. The biodegradability of PLCL was evaluated by subcutaneous implantation in a rat model. The barrier function of the PLCL membrane was investigated in a rat calvaria defect model and compared with commercially available membranes composed of type I collagen (Col) and poly(lactic-co-glycolic acid) (PLGA). An alveolar bone defect model in beagle dogs was used to simulate GBR protocols to evaluate the bone regeneration ability of the experimental PLCL membrane. The PLCL membrane showed slow biodegradation, resulting in an efficient and prolonged barrier function compared with commercial materials. In turn, this barrier function enabled the space-making ability of PLCL membrane and facilitated bone regeneration. In the alveolar bone defect model, significantly greater regeneration was achieved by treatment with PLCL membrane compared with Col and PLGA membranes. Additionally, a continuous alveolar ridge contour was observed in PLCL-treated bone defects. In conclusion, the PLCL bilayer membrane is a promising biomaterial for use in GBR given its slow degradation and prolonged barrier function.

Vascularization of a Bone Organoid Using Dental Pulp Stem Cells.

Bone organoids offer a novel path for the reconstruction and repair of bone defects. We previously fabricated scaffold-free bone organoids using cell constructs comprising only bone marrow-derived mesenchymal stem cells (BMSCs). However, the cells in the millimetre-scale constructs were likely to undergo necrosis because of difficult oxygen diffusion and nutrient delivery. Dental pulp stem cells (DPSCs) are capable of differentiating into vascular endothelial lineages and have great vasculogenic potential under endothelial induction. Therefore, we hypothesized that DPSCs can serve as a vascular source to improve the survival of the BMSCs within the bone organoid. In this study, the DPSCs had greater sprouting ability, and the proangiogenic marker expressions were significantly greater than those of BMSCs. DPSCs were incorporated into the BMSC constructs at various ratios (5%-20%), and their internal structures and vasculogenic and osteogenic characteristics were investigated after endothelial differentiation. As a result, the DPSCs are differentiated into the CD31-positive endothelial lineage in the cell constructs. The incorporation of DPSCs significantly suppressed cell necrosis and improved the viability of the cell constructs. In addition, lumen-like structures were visualized by fluorescently labelled nanoparticles in the DPSC-incorporated cell constructs. The vascularized BMSC constructs were successfully fabricated using the vasculogenic ability of the DPSCs. Next, osteogenic induction was initiated in the vascularized BMSC/DPSC constructs. Compared with only BMSCs, constructs with DPSCs had increased mineralized deposition and a hollow structure. Overall, this study demonstrated that vascularized scaffold-free bone organoids were successfully fabricated by incorporating DPSCs into BMSC constructs, and the biomimetic biomaterial is promising for bone regenerative medicine and drug development.

Intercellular crosstalk in adult dental pulp is mediated by heparin-binding growth factors Pleiotrophin and Midkine.

BACKGROUND: In-depth knowledge of the cellular and molecular composition of dental pulp (DP) and the crosstalk between DP cells that drive tissue homeostasis are not well understood. To address these questions, we performed a comparative analysis of publicly available single-cell transcriptomes of healthy adult human DP to 5 other reference tissues: peripheral blood mononuclear cells, bone marrow, adipose tissue, lung, and skin. RESULTS: Our analysis revealed that DP resident cells have a unique gene expression profile when compared to the reference tissues, and that DP fibroblasts are the main cell type contributing to this expression profile. Genes coding for pleiotrophin (PTN) and midkine (MDK), homologous heparin-binding growth-factors, possessed the highest differential expression levels in DP fibroblasts. In addition, we identified extensive crosstalk between DP fibroblasts and several other DP resident cells, including Schwann cells, mesenchymal stem cells and odontoblasts, mediated by PTN and MDK. CONCLUSIONS: DP fibroblasts emerge as unappreciated players in DP homeostasis, mainly through their crosstalk with glial cells. These findings suggest that fibroblast-derived growth factors possess major regulatory functions and thus have a potential role as dental therapeutic targets.

Fabrication of pH-Responsive Zn2+-Releasing Glass Particles for Smart Antibacterial Restoratives.

The on-demand release of antibacterial components due to pH variations caused by acidogenic/cariogenic bacteria is a possible design for smart antibacterial restorative materials. This study aimed to fabricate pH-responsive Zn2+-releasing glass particles and evaluate their solubilities, ion-releasing characteristics, and antibacterial properties in vitro. Three kinds of silicate-based glass particles containing different molar ratios of Zn (PG-1: 25.3; PG-2: 34.6; PG-3: 42.7 mol%) were fabricated. Each particle was immersed in a pH-adjusted medium, and the solubility and concentration of the released ions were determined. To evaluate the antibacterial effect, Streptococcus mutans was cultured in the pH-adjusted medium in the presence of each particle, and the bacterial number was counted. The solubility and concentration of Zn2+ released in the medium increased with a decrease in medium pH. PG-3 with a greater content of Zn demonstrated higher concentrations of released Zn2+ compared with PG-1 and PG-2. PG-2 exhibited bactericidal effects at pH 5.1, whereas PG-3 demonstrated bactericidal effects at pH values of 5.1 and 6.1, indicating that PG-3 was effective at inhibiting S. mutans even under slightly acidic conditions. The glass particle with 42.7 mol% Zn may be useful for developing smart antibacterial restoratives that contribute to the prevention of diseases such as caries on root surfaces with lower acid resistance.

Large three-dimensional cell constructs for tissue engineering.

Much research has been conducted on fabricating biomimetic biomaterials in vitro. Tissue engineering approaches are often conducted by combining cells, scaffolds, and growth factors. However, the degradation rate of scaffolds is difficult to control and the degradation byproducts occasionally limit tissue regeneration. To overcome these issues, we have developed a novel system using a thermo-responsive hydrogel that forms scaffold-free, three-dimensional (3D) cell constructs with arbitrary size and morphology. 3D cell constructs prepared using bone marrow-derived stromal stem cells (BMSCs) exhibited self-organizing ability and formed bone-like tissue with endochondral ossification. Endothelial cells were then introduced into the BMSC construct and a vessel-like structure was formed within the constructs. Additionally, the bone formation ability was promoted by endothelial cells and cell constructs could be freeze-dried to improve their clinical application. A pre-treatment with specific protein protectant allowed for the fabrication of novel bone substitutes composed only of cells. This 3D cell construct technology using thermo-responsive hydrogels was then applied to other cell species. Cell constructs composed of dental pulp stem cells were fabricated, and the resulting construct regenerated pulp-like tissue within a human pulpless tooth. In this review, we demonstrate the approaches for the in vitro fabrication of bone and dental pulp-like tissue using thermo-responsive hydrogels and their potential applications.

Barrier membranes for tissue regeneration in dentistry.

Background: In dentistry, barrier membranes are used for guided tissue regeneration (GTR) and guided bone regeneration (GBR). Various membranes are commercially available and extensive research and development of novel membranes have been conducted. In general, membranes are required to provide barrier function, biosafety, biocompatibility and appropriate mechanical properties. In addition, membranes are expected to be bioactive to promote tissue regeneration. Objectives: This review aims to organize the fundamental characteristics of the barrier membranes that are available and studied for dentistry, based on their components. Results: The principal components of barrier membranes are divided into nonbiodegradable and biodegradable materials. Nonbiodegradable membranes are manufactured from synthetic polymers, metals or composites of these materials. The first reported barrier membrane was made from expanded polytetrafluoroethylene (e-PTFE). Titanium has also been applied for dental regenerative therapy and shows favorable barrier function. Biodegradable membranes are mainly made from natural and synthetic polymers. Collagens are popular materials that are processed for clinical use by cross-linking. Aliphatic polyesters and their copolymers have been relatively recently introduced into GTR and GBR treatments. In addition, to improve the tissue regenerative function and mechanical strength of biodegradable membranes, inorganic materials such as calcium phosphate and bioactive glass have been incorporated at the research stage. Conclusions: Currently, there are still insufficient guidelines for barrier membrane choice in GTR and GBR, therefore dentists are required to understand the characteristics of barrier membranes.

Fabrication of novel poly(lactic acid/caprolactone) bilayer membrane for GBR application.

OBJECTIVE: Guided bone regeneration (GBR) often involves the use of membranes as barriers for soft tissues. Commercially available membranes, however, do not possess an adequately low degradation rate, resulting in limited barrier function. The purpose of this study was to develop and assess the physicochemical and biological characteristics of a novel poly(l-lactic acid/caprolactone) (PLCL) bilayer membrane and determine its usefulness for GBR application. METHODS: The experimental bilayer membrane was prepared via a two-step freezing and lyophilization process with a PLCL solution. Next, the PLCL membrane was investigated regarding tensile strength, surface roughness, in vitro degradation and clinical operability. In addition, cell proliferation and differentiation were investigated on each layer of the experimental membrane. For all experiments, a commercially available poly(lactic-co-glycolic) acid membrane was used as a control. RESULTS: In vitro analysis of the PLCL bilayer membrane revealed suitable mechanical strength combined with high breaking strain, which contributed to membrane operability. In addition, the PLCL bilayer membrane had enhanced stability compared to the commercial control due to its slower degradation, and was capable of supporting cell growth and osteogenic differentiation. SIGNIFICANCE: The current study confirmed that the PLCL membrane possessed a high biocompatibility and slow degradation rate that contributes to prolonged barrier function and bone regeneration. Altogether, it was considered that the PLCL bilayer membrane developed in this study was applicable for GBR treatment.

Fabrication of strontium-releasable inorganic cement by incorporation of bioactive glass.

OBJECTIVES: Bioactive glass (BG) is widely used as a bioactive material for various clinical applications, and effective and efficient elemental release and an increase in mechanical strength are expected with further development. The purpose of this study is to clarify the physicochemical and biological characteristics of Sr-doped BG-incorporated glass ionomer cements. METHODS: Sr-doped BGs (45SiO2-6P2O5-24.5Na2O-(24.5-x)CaO-xSrO) (wt%), where × = 0, 6, 12, were prepared, and the particle size, crystallinity, and elemental release profiles were evaluated. The Sr-doped BGs were then incorporated into a glass ionomer cement at a weight ratio of 1:4, and the physicochemical properties (compressive strength, bending strength, hardness, and elemental release profile) were investigated. Cell attachment, cell proliferation, and osteoblastic differentiation were used to evaluate the biological characteristics. RESULTS: The Sr-doped BGs were amorphous phases with a homogeneous particle size and exhibited sustained release of Ca, Si, and Sr. The BG-incorporated cements were able to release these elements while retaining the same mechanical properties as those of the pure glass ionomer cement. In addition, no cytotoxicity of osteoblasts or differences in the cell attachment or proliferation were observed for the BG-incorporated cements. In contrast, the Sr-doped BG-incorporated cements promoted the alkaline phosphatase activities of the osteoblasts without the need for any media supplements for osteoblastic differentiation. SIGNIFICANCE: Sr-releasable inorganic cements with high mechanical properties were successfully fabricated by incorporating Sr-doped BGs in glass ionomer cement. These bioactive materials are promising candidates for bone grafting materials, bone cements, and pulp capping materials.

Fabricating large-scale three-dimensional constructs with living cells by processing with syringe needles.

Three-dimensional (3D) cell constructs composed only of cells and cell-secreted extracellular matrix have been attractive biomaterials for tissue engineering technology; however, controlling construct morphology and eliminating dead cells after fabrication remain a challenge. It has been hypothesized that moderate stress could shape constructs and eliminate dead cells. The purpose of this study was to establish an easily available technology for shaping 3D cell constructs and eliminating dead cells postfabrication. To achieve these objectives, spherical cell constructs composed of L-929 fibroblasts were processed using different sized syringe needles. Our results revealed that large-scale rod-shaped cell constructs could be fabricated, and that their diameters could be controlled according to the size of the syringe needle. Additionally, cell viability assays showed that >94% of cells in the rod-shaped constructs were viable, suggesting that dead cells, which have low adhesion force, were dispersed when compressive stress was applied during passage through the needle. The technology described in this study will be promising for future tissue engineering, especially for fabricating elongated tissues such as nerves and blood vessels. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 904-909, 2019.

Photobiomodulation therapy and vitamin C on longevity of cell sheets of human dental pulp stem cells.

Photobiomodulation therapy (PBMT) can improve processes relevant to tissue regeneration, such as survival, proliferation, migration, and differentiation of cells, including stem cells. Thus, PBMT could be applied as auxiliary therapy for tissue regeneration. Cell sheets (CSs) induced by vitamin C (VC) can generate large amount of cells, which would also be useful for tissue regeneration. VC and PBMT cause opposite effects on cell metabolism (e.g., VC is antioxidative, and PBMT generates reactive oxygen species); however, hDPSC CSs were formed when VC and PBMT+VC were applied. Thus, this study showed that PBMT does not interfere with the formation of cell sheets induced by VC. Additionally, PBMT improved the functional differentiation of the cells isolated from the CSs. Thus, due to the clinical benefits of PBMT, the association of this therapy with cell sheets seems promising for future applications in tissue regeneration.