Direct contact with endothelial cells drives dental pulp stem cells toward smooth muscle cells differentiation via TGF-ß1 secretion.

AIM: Prevascularization is vital to accelerate functional blood circulation establishment in transplanted engineered tissue constructs. Mesenchymal stem cells (MSCs) or mural cells could promote the survival of implanted endothelial cells (ECs) and enhance the stabilization of newly formed blood vessels. However, the dynamic cell-cell interactions between MSCs, mural cells and ECs in the angiogenic processes remain unclear. This study aimed to explore the interactions of human umbilical vein ECs (HUVECs) and dental pulp stem cells (DPSCs) in an in vitro cell coculture model. METHODOLOGY: Human umbilical vascular ECs and DPSCs were directly cocultured or indirectly cocultured with transwell inserts in endothelial basal media-2 (EBM-2) supplemented with 5% FBS for 6 days. Expression of SMC-specific markers in DPSCs monoculture and HUVEC+DPSC cocultures was assessed by western blot and immunofluorescence. Activin A and transforming growth factor-beta 1 (TGF-ß1) in conditioned media (CM) of HUVECs monoculture (E-CM), DPSCs monoculture (D-CM) and HUVEC+DPSC cocultures (E+D-CM) were analysed by enzyme-linked immunosorbent assay. TGF-ß RI kinase inhibitor VI, SB431542, was used to block TGF-ß1/ALK5 signalling in DPSCs. RESULTS: The expression of SMC-specific markers, α-SMA, SM22α and Calponin, were markedly increased in HUVEC+DPSC direct cocultures compared to that in DPSCs monoculture, while no differences were demonstrated between HUVEC+DPSC indirect cocultures and DPSCs monoculture. E+D-CM significantly upregulated the expression of SMC-specific markers in DPSCs compared to E-CM and D-CM. Activin A and TGF-ß1 were considerably higher in E+D-CM than that in D-CM, with upregulated Smad2 phosphorylation in HUVEC+DPSC cocultures. Treatment with activin A did not change the expression of SMC-specific markers in DPSCs, while treatment with TGF-ß1 significantly enhanced these markers' expression in DPSCs. In addition, blocking TGF-ß1/ALK5 signalling inhibited the expression of α-SMA, SM22α and Calponin in DPSCs. CONCLUSIONS: TGF-ß1 was responsible for DPSC differentiation into SMCs in HUVEC+DPSC cocultures, and TGF-ß1/ALK5 signalling pathway played a vital role in this process.

DPSCs treated by TGF-ß1 regulate angiogenic sprouting of three-dimensionally co-cultured HUVECs and DPSCs through VEGF-Ang-Tie2 signaling.

BACKGROUND: Maintaining the stability and maturation of blood vessels is of paramount importance for the vessels to carry out their physiological function. Smooth muscle cells (SMCs), pericytes, and mesenchymal stem cells (MSCs) are involved in the maturation process of the newly formed vessels. The aim of this study was to investigate whether transforming growth factor beta 1 (TGF-ß1) treatment could enhance pericyte-like properties of dental pulp stem cells (DPSCs) and how TGF-ß1-treated DPSCs for 7 days (T-DPSCs) stabilize the newly formed blood vessels. METHODS: We utilized TGF-ß1 to treat DPSCs for 1, 3, 5, and 7 days. Western blotting and immunofluorescence were used to analyze the expression of SMC markers. Functional contraction assay was conducted to assess the contractility of T-DPSCs. The effects of T-DPSC-conditioned media (T-DPSC-CM) on human umbilical vein endothelial cell (HUVEC) proliferation and migration were examined by MTT, wound healing, and trans-well migration assay. Most importantly, in vitro 3D co-culture spheroidal sprouting assay was used to investigate the regulating role of vascular endothelial growth factor (VEGF)-angiopoietin (Ang)-Tie2 signaling on angiogenic sprouting in 3D co-cultured spheroids of HUVECs and T-DPSCs. Angiopoietin 2 (Ang2) and VEGF were used to treat the co-cultured spheroids to explore their roles in angiogenic sprouting. Inhibitors for Tie2 and VEGFR2 were used to block Ang1/Tie2 and VFGF/VEGFR2 signaling. RESULTS: Western blotting and immunofluorescence showed that the expression of SMC-specific markers (α-SMA and SM22α) were significantly increased after treatment with TGF-ß1. Contractility of T-DPSCs was greater compared with that of DPSCs. T-DPSC-CM inhibited HUVEC migration. In vitro sprouting assay demonstrated that T-DPSCs enclosed HUVECs, resembling pericyte-like cells. Compared to co-culture with DPSCs, a smaller number of HUVEC sprouting was observed when co-cultured with T-DPSCs. VEGF and Ang2 co-stimulation significantly enhanced sprouting in HUVEC and T-DPSC co-culture spheroids, whereas VEGF or Ang2 alone exerted insignificant effects on HUVEC sprouting. Blocking Tie2 signaling reversed the sprouting inhibition by T-DPSCs, while blocking VEGF receptor (VEGFR) signaling boosted the sprouting inhibition by T-DPSCs. CONCLUSIONS: This study revealed that TGF-ß1 can induce DPSC differentiation into functional pericyte-like cells. T-DPSCs maintain vessel stability through Ang1/Tie2 and VEGF/VEGFR2 signaling.

Fabrication of Tapered Fluidic Microchannels Conducive to Angiogenic Sprouting within Gelatin Methacryloyl Hydrogels.

INTRODUCTION: The transplantation of stem cells/tissue constructs into root canal space is a promising strategy for regenerating lost pulp tissue. However, the root canal system, which is cone shaped with a taper from the larger coronal end to the smaller apical end, limits the vascular supply and, therefore, the regenerative capacity. The current study aimed to fabricate built-in microchannels with different tapers to explore various approaches to endothelialize these microchannels. METHODS: The fluidic microchannels with varying tapers (parallel, 0.04, and 0.06) were fabricated within gelatin methacryloyl (GelMA) hydrogel (with or without stem cell from the apical papilla [SCAP] encapsulation) of different concentrations (5%, 7.5%, and 10% [w/v]). Green fluorescent protein-expressing human umbilical vein endothelial cells (HUVECs-GFP) were seeded alone or with SCAPs in coculture into these microchannels. Angiogenic sprouting was assessed by fluorescence and a confocal microscope and ImageJ software (National Institutes of Health, Bethesda, MD). Immunostaining was conducted to illustrate monolayer formation. Data were statistically analyzed by 1-way/2-way analysis of variance. RESULTS: HUVEC-only inoculation formed an endothelial monolayer inside the microchannel without angiogenic sprouting. HUVECs-GFP/SCAPs cocultured at a 1:1 ratio produced the longest sprouting compared with the other 3 ratios. The average length of the sprouting in the 0.04 taper microchannel was significantly longer compared with that in the parallel and 0.06 taper microchannels. Significant differences in HUVEC-GFP sprouting were observed in 5% GelMA hydrogel. Encapsulation of SCAPs within hydrogel further stimulated the sprouting of HUVECs. CONCLUSIONS: The coculture of SCAPs and HUVECs-GFP at a ratio of 1:1 in 0.04 taper fluidic microchannels fabricated with 5% (w/v) GelMA hydrogel with SCAPs encapsulated was found to be the optimal condition to enhance angiogenesis inside tapered microchannels.