Fibrous Topography-Potentiated Canonical Wnt Signaling Directs the Odontoblastic Differentiation of Dental Pulp-Derived Stem Cells.

Nanofibrous engineered matrices have significant potential in cellular differentiation and tissue regeneration. Stem cells require specific extracellular signals that lead to the induction of different lineages. However, the mechanisms by which the nanofibrous matrix promotes mesenchymal stem cell (MSC) differentiation are largely unknown. Here, we investigated the mechanisms that underlie nanofibrous matrix-induced odontoblastic differentiation of human dental pulp MSCs (DP-MSCs). An electrospun polystyrene nanofibrous (PSF) matrix was prepared, and the cell responses to the PSF matrix were assessed in comparison with those on conventional tissue culture dishes. The PSF matrix promoted the expression of Wnt3a, Wnt5a, Wnt10a, BMP2, BMP4, and BMP7 in the DP-MSCs, concomitant with the induction of odontoblast/osteoblast differentiation markers, dentin sialophosphoprotein (DSPP), osteocalcin, and bone sialoprotein, whose levels were further enhanced by treatment with recombinant Wnt3a. The DP-MSCs cultured on the PSF matrix also exhibited a high alkaline phosphatase activity and intense Alizarin Red staining, indicating that the PSF matrix promotes odontoblast differentiation. Besides inducing the expression of Wnt3a, the PSF matrix maintained high levels of ß-catenin protein and enhanced its translocation to the nucleus, leading to its transcriptional activity. Forced expression of LEF1 or treatments with LiCl further enhanced the DSPP expression. Blocking the Wnt3a-initiated signaling abrogated the PSF-induced DSPP expression. Furthermore, the cells on the PSF matrix increased the DSPP promoter activity. The ß-catenin complex was bound to the conserved motifs on the DSPP promoter dictating its transcription. Transplantations of the preodontoblast-seeded PSF matrix to the subcutaneous tissues of nude mice confirmed the association of the PSF matrix with the Wnt3a and DSPP expressions in vivo. Taken together, these results demonstrate the nanofibrous engineered matrix strongly supports odontoblastic differentiation of DP-MSCs by enhancing Wnt/ß-catenin signaling.

Dimethyloxalylglycine-embedded Poly(ε-caprolactone) Fiber Meshes Promote Odontoblastic Differentiation of Human Dental Pulp-derived Cells.

INTRODUCTION: The in vivo effect of prolyl hydroxylase inhibitors on the regeneration of the pulp-dentin complex is unclear. The purpose of this study was to investigate the effect of dimethyloxalylglycine (DMOG)-embedded poly(ε-caprolactone) fiber (PCLF/DMOG) on odontoblastic differentiation of human dental pulp-derived cells (hDPCs) by transplantation of the dentin slice model. METHODS: The hDPCs were seeded onto electrospun PCLF and PCLF/DMOG in dentin slices and then transplanted into nude mice. The surface topography was evaluated for both PCLFs, and DMOG release from the PCLF/DMOG was examined. The effects of the PCLF/DMOG were assessed by histology and quantitative reverse transcription polymerase chain reaction. RESULTS: The PCLF/DMOG-treated dentin slices showed higher cellularity with a palisading arrangement of hDPCs and organized collagen fibers. We found that the PCLF/DMOG significantly stimulated the expression of vascular endothelial growth factor, dentin sialoprotein, and bone sialoprotein in the hDPCs (P < .05) and mouse vascular endothelial growth factor A, mouse platelet endothelial cell adhesion molecule 1, and mouse neurofilament light polypeptide in the surrounding host cells (P < .05). CONCLUSIONS: These results show that PCLF/DMOG has potential in pulp-dentin complex regeneration by promoting odontoblastic differentiation of hDPCs and by enhancing host cell recruitment, angiogenesis, and neurogenesis through the released DMOG-mediated cell responses.

Effects of the fibrous topography-mediated macrophage phenotype transition on the recruitment of mesenchymal stem cells: An in vivo study.

Host responses to a biomaterial critically influence its in vivo performance. Biomaterial architectures that can recruit endogenous host stem cells could be beneficial in tissue regeneration or integration. Here, we report that the fibrous topography of biomaterials promotes the recruitment of host mesenchymal stem cells (MSCs) by facilitating the macrophage phenotype transition from M1-to-M2. Electrospun poly (ε-caprolactone) fiber (PCL-fiber) films were implanted into the subcutaneous tissues of rats, and the response of host cells to the PCL-fiber was evaluated and compared with those of solid ones (PCL-solid). During the initial post-implantation period, greater numbers of cells were recruited and adhered to the PCL-fiber compared to the PCL-solid, and the cells exhibited the M1 phenotype, which was supported by the enhanced adsorption of complement C3a to the implanted PCL-fiber. Subsequently, the PCL-fiber supported the macrophage phenotype transition from M1-to-M2, which was confirmed by the ratio of M2/M1 marker (CD163/CCR7)-positive cells and by the expression of M2/M1 markers (arginase-1/iNOS). The PCL-fiber also reduced the formation of foreign body giant cells. MSC marker (CD29, CD44, and CD90)-positive cells began to appear as early as day 4 on the PCL-fiber, while few MSCs were observed on the PCL-solid. The MSCs migration ex vivo assay showed that MSCs substantially migrated across the trans-wells toward the implanted PCL-fiber. The cells on the implanted PCL-fiber expressed and secreted substantial levels of SDF-1 (CXCL-12), while anti-SDF-1 neutralizing antibody abrogated the MSCs migration. Taken together, these results provide evidence that the fibrous topography of biomaterials enhances the recruitment of MSCs by promoting macrophage recruitment, facilitating M1-to-M2 transition, and enhancing SDF-1 secretion.

Effects of the incorporation of ε-aminocaproic acid/chitosan particles to fibrin on cementoblast differentiation and cementum regeneration.

Cementum formation on the exposed tooth-root surface is a critical process in periodontal regeneration. Although various therapeutic approaches have been developed, regeneration of integrated and functional periodontal complexes is still wanting. Here, we found that the OCCM30 cementoblasts cultured on fibrin matrix express substantial levels of matrix proteinases, leading to the degradation of fibrin and the apoptosis of OCCM30 cells, which was reversed upon treatment with a proteinase inhibitor, ε-aminocaproic acid (ACA). Based on these findings, ACA-releasing chitosan particles (ACP) were fabricated and ACP-incorporated fibrin (fibrin-ACP) promoted the differentiation of cementoblasts in vitro, as confirmed by bio-mineralization and expressions of molecules associated with mineralization. In a periodontal defect model of beagle dogs, fibrin-ACP resulted in substantial cementum formation on the exposed root dentin in vivo, compared to fibrin-only and enamel matrix derivative (EMD) which is used clinically for periodontal regeneration. Remarkably, the fibrin-ACP developed structural integrations of the cementum-periodontal ligament-bone complex by the Sharpey's fiber insertion. In addition, fibrin-ACP promoted alveolar bone regeneration through increased bone volume of tooth roof-of-furcation defects and root coverage. Therefore, fibrin-ACP can promote cementogenesis and osteogenesis by controlling biodegradability of fibrin, implicating the feasibility of its therapeutic use to improve periodontal regeneration. STATEMENT OF SIGNIFICANCE: Cementum, the mineralized layer on root dentin surfaces, functions to anchor fibrous connective tissues on tooth-root surfaces with the collagenous Sharpey's fibers integration, of which are essential for periodontal functioning restoration in the complex. Through the cementum-responsible fiber insertions on tooth-root surfaces, PDLs transmit various mechanical responses to periodontal complexes against masticatory/occlusal stimulations to support teeth. In this study, periodontal tissue regeneration was enhanced by use of modified fibrin biomaterial which significantly promoted cementogenesis within the periodontal complex with structural integration by collagenous Sharpey's fiber insertions in vivo by controlling fibrin degradation and consequent cementoblast apoptosis. Furthermore, the modified fibrin could improve repair and regeneration of tooth roof-of-furcation defects, which has spatial curvatures and geometrical difficulties and hardly regenerates periodontal tissues.

Effects of Dimethyloxalylglycine-Embedded Poly(ε-caprolactone) Fiber Meshes on Wound Healing in Diabetic Rats.

Impaired wound healing in diabetic patients is associated with altered inflammatory responses, poor angiogenesis, deficient extracellular matrix (ECM) component, and peripheral neuropathy. To develop a wound dressing that is capable of the controlled delivery of bioactive small molecules that can improve diabetic wound healing, dimethyloxalylglycine (DMOG)-embedded poly(ε-caprolactone) (PCL) fiber (PCLF/DMOG) meshes are fabricated by electrospinning, and the effects of the PCLF/DMOG meshes on wound healing in diabetic rats are evaluated. Electrospun PCLF/DMOG meshes increase not only the wound closure, re-epithelialization ratio, epithelial maturation (K-10-positive epidermis), and collagen-positive area but also the numbers of angiogenic marker (CD-31)-positive and neuronal marker (neurofilament)-positive cells compared to PCLF (p < 0.05). In in vitro examinations, RAW264.7 macrophages grown on PCLF/DMOG meshes enhance the expression of growth factors (IGF-1, HB-EGF, and NGF) and anti-inflammatory factors (TGF-ß1 and IL-4) but decrease that of pro-inflammatory factors (IL-1ß and IL-6). Keratinocyte migration is increased by conditioned media from the cultures of the macrophages grown either in the presence of DMOG or on PCLF/DMOG. Collectively, these results indicate that PCLF/DMOG meshes promote impaired wound healing in diabetic rats by modulating macrophage responses, enhancing angiogenesis and nerve innervation, and improving ECM synthesis.

Regenerative Characteristics of Apical Papilla-derived Cells from Immature Teeth with Pulpal and Periapical Pathosis.

INTRODUCTION: The purpose of this study was to ascertain the regenerative characteristics of apical papilla-derived cells (APDCs) from immature teeth with pulpal and periapical pathosis and thus to provide proof-of-principle evidence for further regenerative endodontic research. METHODS: Pulpal and periapical pathosis was induced in immature permanent double-rooted premolars of beagles, which were randomly assigned to experimental treatment groups: group AO (n = 14), pulp disruption and access left open; group PS (n = 14), supragingival plaque suspension-soaked cotton pellet was introduced, and access was sealed; and control (n = 7), untreated. The teeth were extracted at 2- and 4-week periods after experimental treatments. APDCs were cultured from the extracted teeth, and their cellular proliferation, differentiation characteristics, and stemness were assessed. The data were statistically analyzed. RESULTS: After 4 weeks of intentional pulpal and periapical pathosis induction period, all teeth in group PS showed features of apical periodontitis with necrotic pulp, and their APDCs showed significantly increased proliferation rate and osteogenic/odontogenic differentiation capabilities (P < .05). The stemness was maintained in all APDCs, although the stem cell population was smaller in group PS at 2-week period when the inflammatory responses were most fulminant (P < .05). CONCLUSIONS: The APDCs from immature teeth retained the regenerative characteristics with the differences according to their pulpal and periapical pathosis. The results of this study partly provide the evidence for regenerative endodontic research.

Compressive Behavior of Fiber-Reinforced Concrete with End-Hooked Steel Fibers.

In this paper, the compressive behavior of fiber-reinforced concrete with end-hooked steel fibers has been investigated through a uniaxial compression test in which the variables were concrete compressive strength, fiber volumetric ratio, and fiber aspect ratio (length to diameter). In order to minimize the effect of specimen size on fiber distribution, 48 cylinder specimens 150 mm in diameter and 300 mm in height were prepared and then subjected to uniaxial compression. From the test results, it was shown that steel fiber-reinforced concrete (SFRC) specimens exhibited ductile behavior after reaching their compressive strength. It was also shown that the strain at the compressive strength generally increased along with an increase in the fiber volumetric ratio and fiber aspect ratio, while the elastic modulus decreased. With consideration for the effect of steel fibers, a model for the stress-strain relationship of SFRC under compression is proposed here. Simple formulae to predict the strain at the compressive strength and the elastic modulus of SFRC were developed as well. The proposed model and formulae will be useful for realistic predictions of the structural behavior of SFRC members or structures.

Synergistic effects of dimethyloxalylglycine and butyrate incorporated into α-calcium sulfate on bone regeneration.

Osteogenesis is closely related to angiogenesis, and the combined delivery of angiogenic and osteogenic factors has been suggested to enhance bone regeneration. Small molecules have been explored as alternatives to growth factors for tissue regeneration applications. In this study, we examined the effects of the combined application of angiogenic and osteogenic small molecules on bone regeneration using a prolyl hydroxylase, dimethyloxalylglycine (DMOG), and a histone deacetylase inhibitor, butyrate. In a critical size bone defect model in rats, DMOG and butyrate, which were incorporated into α calcium sulfate (αCS), resulted in synergistic enhancements in bone and blood vessel formation, eventually leading to bone healing, as confirmed by micro-CT and histological analyses. In MC4 pre-osteoblast cultures, DMOG and butyrate enhanced the pro-angiogenic responses and osteoblast differentiation, respectively, which were evaluated based on the levels of hypoxia inducible factor (HIF)-1α protein and the expression of pro-angiogenic molecules (VEGF, home oxidase-1, glucose transporter-1) and by alkaline phosphatase (ALP) activity and the expression of osteoblast phenotype marker molecules (ALP, α1(I)col, osteocalcin, and bone sialoprotein). DMOG combined with butyrate synergistically improved osteoblast differentiation and pro-angiogenic responses, the levels of which were drastically increased in the cultures on αCS disks. Furthermore, it was demonstrated that αCS increased the level of HIF-1α and as a consequence VEGF expression, and supported osteoblast differentiation through the release of calcium ions from the αCS. Altogether, the results of this study provide evidence that a combination treatment with the small molecules DMOG and butyrate can expedite the process of bone regeneration and that αCS can be an efficient delivery vehicle for the small molecules for bone regeneration.

Comparative evaluation of the biological properties of fibrin for bone regeneration.

Fibrin is a natural provisional matrix found in wound healing, while type I collagen is a major organic component of bone matrix. Despite the frequent use of fibrin and type I collagen in bone regenerative approaches, their comparative efficacies have not yet been evaluated. In the present study, we compared the effects of fibrin and collagen on the proliferation and differentiation of osteoblasts and protein adsorption. Compared to collagen, fibrin adsorbed approximately 6.7 times more serum fibronectin. Moreover, fibrin allowed the proliferation of larger MC3T3-E1 pre-osteoblasts, especially at a low cell density. Fibrin promoted osteoblast differentiation at higher levels than collagen, as confirmed by Runx2 expression and transcriptional activity, alkaline phosphatase activity, and calcium deposition. The results of the present study suggest that fibrin is superior to collagen in the support of bone regeneration.

Analysis of histone deacetylase inhibitor-induced responses in human periodontal ligament fibroblasts.

Periodontal ligament (PDL) fibroblasts play critical roles in the regeneration of periodontal tissues damaged by periodontitis. Histone deacetylase inhibitors (HDIs) have been suggested to be potential tools in tissue engineering. The feasibility of using the HDI, sodium butyrate (NaB) for periodontal regeneration was examined by evaluating its effect on the osteogenic differentiation of human PDL fibroblasts and its modulation of the inflammatory responses to lipopolysaccharide (LPS). NaB did not cause significant cell death at 100 µM but promoted the expression of the osteoblast phenotype (Runx2, osterix, osteocalcin, and bone sialoprotein). NaB significantly inhibited the LPS-induced production of reactive oxygen species and the expression of pro-inflammatory cytokines (IL-1ß and TNF-α). These results suggest that HDIs can offer a potential therapeutic agent for periodontal regeneration.

Performance of electrospun poly(ε-caprolactone) fiber meshes used with mineral trioxide aggregates in a pulp capping procedure.

Living dental pulp tissue exposed to the oral environment should be protected with an appropriate pulp capping material to support the dentinogenesis potential of the pulp cells. Mineral trioxide aggregate (MTA) is the material of choice for the treatment of pulp. However, due to cytotoxicity during the initial setting phase of MTA, a new material is required that can act as a barrier to direct contact but facilitate the favorable effect of MTA. This study examined the feasibility of using electrospun poly(ε-caprolactone) fiber (PCL-F) meshes in the MTA-based pulp capping procedures. An experimental pulp capping was performed on the premolars of beagle dogs, and the efficacy of the PCL-F meshes was evaluated after 8 weeks. PCL-F/MTA formed a dentin bridge that was approximately fourfold thicker than that formed by the MTA. Columnar polarized odontoblast-like cells with long processes and tubular dentin-like matrices were observed beneath the dentin bridge in the PCL-F/MTA. The cells were also intensely immunostained for dentin sialoprotein. In cell cultures, PCL-F/MTA reduced cell death to ~8% of that in the MTA group. The proliferation of the cells cultured on PCL-F/MTA was much greater than that of cells cultured on MTA. Furthermore, PCL-F/MTA promoted the differentiation of MDPC23 cells to odontoblast-like cells and biomineralization, as confirmed by the expression of alkaline phosphatase and dentin sialophosphoprotein, and by the deposition of calcium. Based on these histologic findings and the cell responses observed in this study, PCL-F may be used efficiently in the MTA-based dental pulp therapy.

The effects of the modulation of the fibronectin-binding capacity of fibrin by thrombin on osteoblast differentiation.

Fibrin is a natural provisional matrix involved in wound healing and is widely utilized for tissue regeneration. The biological performance of fibrin is largely dependent on its composition and related structures. In this study, we examined the effect of thrombin, which is engaged with fibrin, on osteoblast differentiation and its mode of action. Fibrin matrices were prepared with different concentrations of thrombin, and MC3T3-E1 pre-osteoblasts were cultured on the fibrin matrices. Thrombin-promoted fibrin-enhanced osteoblast differentiation in a dose-dependent manner, as confirmed by the extent of calcium deposition, alkaline phosphatase activity, and the level of Runx2. The synthetic activating peptide of protease-activated receptor 1 (PAR1), a prototype receptor of thrombin in osteoblast, did not alter the level of Runx2. Instead, the thrombin that was engaged with fibrin in a dose-dependent manner increased the phosphorylation of integrins ß1 and ß3. The integrin-blocking peptide RGDS reduced the thrombin-enhanced Runx2 in the cells grown on fibrin, whereas the non-functional peptide RGES did not change the level of Runx2. Furthermore, thrombin dose-dependently increased the fibronectin-binding of fibrin. The thrombin-induced integrin phosphorylation and Runx2 expression were also attenuated through the use of a blocking peptide to inhibit the binding of fibronectin to fibrin. The results in this study provide evidence that thrombin engaged with fibrin accelerates osteoblast differentiation via integrins but not PAR1 by modulating the fibronectin-binding capacity of fibrin.

Suppression of Runx2 protein degradation by fibrous engineered matrix.

The fibre structure of engineered matrix that mimic the morphology of type I collagen has exhibited good biological performance for bone regeneration. However, the mechanism by which synthetic fibres promote osteoblast differentiation has yet to be determined. In this study, we demonstrate that fibre structure of an engineered matrix suppresses the degradation of Runx2, a master transcription factor that can turn on to osteoblast differentiation. MC3T3-E1 pre-osteoblasts grown on a fibrous collagen matrix sustained a higher level of Runx2 protein than those on tissue culture dishes or on a collagenase-treated, non-fibrous collagen matrix. The ubiquitin-dependent degradation of Runx2 was profoundly decreased in cells grown on the fibrous collagen matrix. The forced expression of Smurf1, an ubiquitin ligase responsible for Runx2 degradation, abrogated the collagen fibre-induced increase of Runx2. We also prepared a polystyrene fibre matrix, and confirmed that the fibre matrix stabilised the Runx2 protein in MC3T3-E1. Furthermore, we genetically modified C2C12 myoblasts with Runx2, cultured the cells on polystyrene fibre matrix, and observed that the fibre matrix stabilised and sustained exogenous Runx2, which led to the promotion of osteoblast differentiation. Our findings in this study provide evidence that the fibre structure of an engineered matrix contributes to osteoblast differentiation by stabilising the Runx2 protein.