Enhancing Vasculogenesis in Dental Pulp Development: DPSCs-ECs Communication via FN1-ITGA5 Signaling.

BACKGROUND: Dental pulp regeneration therapy is a challenge to achieve early vascularization during treatment. Studying the regulatory mechanisms of vascular formation during human dental pulp development may provide insights for related therapies. In this study, we utilized single-cell sequencing analysis to compare the gene expression of dental pulp stem cells (DPSCs) and vascular endothelial cells (ECs) from developing and mature dental pulps. METHOD: Immunohistochemistry, Western blot, and real-time polymerase chain reaction (RT-PCR) were used to detect fibronectin 1 (FN1) expression and molecules, such as PI3K/AKT. Cell proliferation assay, scratch assay, tube formation assay and were used to investigate the effects of DPSCs on the vasculogenetic capability of ECs. Additionally, animal experiments involving mice were conducted. RESULT: The results revealed that DPSCs exist around dental pulp vasculature. FN1 expression was significantly higher in DPSCs from young permanent pulps than mature pulps, promoting HUVEC proliferation, migration, and tube formation via ITGA5 and the downstream PI3K/AKT signaling pathway. CONCLUSION: Our data indicate that intercellular communication between DPSCs and ECs mediated by FN1-ITGA5 signaling is crucial for vascularizationduring dental pulp development, laying an experimental foundation for future clinical studies.

Distinctive Roles of Wnt Signaling in Chondrogenic Differentiation of BMSCs under Coupling of Pressure and Platelet-Rich Fibrin.

BACKGROUND: Although newly formed constructs of feasible pressure-preadjusted bone marrow mesenchymal stem cells (BMSCs) and platelet-rich fibrin (PRF) showed biomechanical flexibility and superior capacity for cartilage regeneration, it is still not very clear how BMSCs and seed cells feel mechanical stimuli and convert them into biological signals, and the difference in signal transduction underlying mechanical and chemical cues is also unclear. METHODS: To determine whether mechanical stimulation (hydrostatic pressure) and chemical cues (platelet-rich fibrin, PRF) activate canonical or noncanonical Wnt signaling in BMSCs, BMSCs cocultured with PRF were subjected to hydrostatic pressure loading, and the activation of the Wnt signaling molecules and expression of cartilage-associated proteins and genes were determined by western blotting and polymerase chain reaction (PCR). Inhibitors of canonical or noncanonical Wnt signaling, XVX-939 or L690,330, were adopted to investigate the role of Wnt signaling molecules in mechanically promoted chondrogenic differentiation of BMSCs. RESULTS: Hydrostatic pressure of 120 kPa activated both Wnt/ß-catenin signaling and Wnt/Ca2+ signaling, with the the maximum promotion effect at 60 min. PRF exerted no synergistic effect on Wnt/ß-catenin signaling activation. However, the growth factors released by PRF might reverse the promotion effects of pressure on Wnt/Ca2+ signaling. Real-time PCR and Western blotting results showed that pressure could activate the expression of Col-II, Sox9, and aggrecan in BMSCs cocultured with PRF. Blocking experiment found a positive role of Wnt/ß-catenin signaling, and a negative role of Wnt/Ca2+ signaling in chondrogenic differentiation of the BMSCs. Mutual inhibition exists between canonical and noncanonical Wnt signaling in BMSCs under pressure. CONCLUSION: Wnt signaling participates in the pressure-promoted chondrogenesis of the BMSCs co-cultured with PRF, with canonical and noncanonical pathways playing distinct roles during the process.

A novel construct with biomechanical flexibility for articular cartilage regeneration.

BACKGROUND: Although tissue-engineered cartilage has been broadly studied, complete integration of regenerated cartilage with residual cartilage is still difficult for the inferior mechanical and biochemical feature of neocartilage. Chondrogenesis of mesenchymal stem cells can be induced by biophysical and biochemical factors. METHODS: In this study, autologous platelet-rich fibrin (PRF) membrane was used as a growth factor-rich scaffold that may facilitate differentiation of the transplanted bone marrow mesenchymal stem cells (BMSCs). At the same time, hydrostatic pressure was adopted for pre-adjustment of the seed cells before transplantation that may promote the mechanical flexibility of neocartilage. RESULTS: An in vitro study showed that the feasible hydrostatic pressure stimulation substantially promoted the chondrogenic potential of in vitro-cultured BMSC/PRF construct. In vivo results revealed that at every time point, the newborn tissues were the most favorable in the pressure-pretreated BMSC/PRF transplant group. Besides, the transplantation of feasible hydrostatic pressure-pretreated construct by BMSC sheet fragments and PRF granules could obviously improve the integration between the regenerated cartilage and host cartilage milieu, and thereby achieve boundaryless repair between the neocartilage and residual host cartilage tissue in rabbit temporomandibular joints. It could be concluded that feasible hydrostatic pressure may effectively promote the proliferation and chondrogenic differentiation of BMSCs in a BMSC/PRF construct. CONCLUSION: This newly formed construct with biomechanical flexibility showed a superior capacity for cartilage regeneration by promoting the mechanical properties and integration of neocartilage.

The role of anthrax toxin protein receptor 1 as a new mechanosensor molecule and its mechanotransduction in BMSCs under hydrostatic pressure.

Anthrax toxin protein receptor (ANTXR) 1 has many similarities to integrin and is regarded in some respects as a single-stranded integrin protein. However, it is not clear whether ANTXR1 responds to mechanical signals secondary to the activation of integrins or whether it is a completely new, independent and previously undiscovered mechanosensor that responds to an undefined subset of mechanical signaling molecules. Our study demonstrates that ANTXR1 is a novel mechanosensor on the cell membrane, acting independently from the classical mechanoreceptor molecule integrinß1. We show that bone marrow stromal cells (BMSCs) respond to the hydrostatic pressure towards chondrogenic differentiation partly through the glycogen synthase kinase (GSK) 3ß/ß-Catenin signaling pathway, which can be partly regulated by ANTXR1 and might be related to the direct binding between ANTXR1 and low-density lipoprotein receptor-related protein (LRP) 5/6. In addition, ANTXR1 specifically activates Smad2 and upregulates Smad4 expression to facilitate the transport of activated Smad2 to the nucleus to regulate chondrogenesis, which might be related to the direct binding between ANTXR1 and Actin/Fascin1. We also demonstrate that ANTXR1 binds to some extent with integrinß1, but this interaction does not affect the expression and function of either protein under pressure. Thus, we conclude that ANTXR1 plays a crucial role in BMSC mechanotransduction and controls specific signaling pathways that are distinct from those of integrin to influence the chondrogenic responses of BMSCs under hydrostatic pressure.

Improvement of the cytocompatibility of electrospun poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] mats by Ecoflex.

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (PHBV) is a nature-derived polyester with potential application in tissue engineering scaffolds. However, PHBV is associated with disadvantages including high brittleness, slow degradation, high hydrophobicity, and unsatisfactory biocompatibility. In this study, we sought to improve the properties of PHBV by blending it with Ecoflex, a synthetic biopolyester with a high flexibility, fast degradation, and comparatively higher hydrophilicity. PHBV was codissolved with Ecoflex in dichloromethane at different mass ratios (PHBV/Ecoflex: 100/0, 70/30, 50/50, and 30/70) and electrospun into mats. Compared with the pure PHBV mat, the Ecoflex-containing mats showed decreased contact angles with phosphate-buffered saline (PBS), accelerated weight loss in PBS, and increased strain at break with increasing Ecoflex mass ratios. In vitro cell culture also showed significantly improved adhesion and proliferation of human bone marrow stroma cells with the introduction of Ecoflex. Blending PHBV with Ecoflex is a simple and effective method to improve the chemical, mechanical, and biological properties of PHBV simultaneously and thereby to expedite its application in tissue engineering. To our knowledge, this is the first report showing the biocompatibility of Ecoflex-containing materials with human cells.

Psychological stress delays periodontitis healing in rats: the involvement of basic fibroblast growth factor.

OBJECTIVE: To evaluate the effects of psychological stress on periodontitis healing in rats and the contribution of basic fibroblast growth factor (bFGF) expression to the healing process. METHODS: Ninety-six rats were randomly distributed into control group, periodontitis group, and periodontitis plus stress group. Then, the rats were sacrificed at baseline and week(s) 1, 2, and 4. The periodontitis healing condition was assessed, and the expression of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and bFGF were tested by immunohistochemistry. RESULTS: The stressed rats showed reduced body weight gain, behavioral changes, and increased serum corticosterone and ACTH levels (P < 0.05). The surface of inflammatory infiltrate, alveolar bone loss, attachment loss, and expression of IL-1ß and TNF-α in the stress group were higher than those in the periodontitis group at weeks 2 and 4 (P < 0.05). Rats with experimental periodontitis showed decreased bFGF expression (P < 0.05), and the recovery of bFGF expression in the stress group was slower than that in the periodontitis group (P < 0.05). Negative correlations between inflammatory cytokines and bFGF were detected. CONCLUSION: Psychological stress could delay periodontitis healing in rats, which may be partly mediated by downregulation of the expression of bFGF in the periodontal ligament.