Application of microfluidic platform in promoting angiogenesis by co-culture of stem cells from the apical papilla and endothelial cells in gelatin methacryloyl hydrogel

Abstract

Regeneration of dental pulp is one of the most promising therapeutic strategies for dental pulp infection and necrosis. However, the dental pulp tissue within the root canal system is encased by impermeable dentin. Furthermore, the root canal system has no collateral blood supply except one root apical opening to obtain blood circulation. These unique anatomies limit the regeneration potential of the current regenerative approaches and pose a significant challenge for pulp tissue regeneration. Therefore, this study fabricated three-dimensional (3D) pre-vascular networks in GelMA hydrogel to enhance angiogenesis and pulp/dentin regeneration. One of the viable engineering techniques to encourage angiogenesis is fabricating the built-in fluidic vascular microchannels in the middle of GelMA hydrogel. GelMA hydrogel can offer a proper milieu for developing perfusable microvascular networks of various architectural configurations, and the built-in microchannels can enhance cell survival and differentiation. Therefore, in the first part of this study, we investigated whether the varying concentrations of GelMA hydrogel scaffold in combination with the built-in fluidic vascular channels with different tapers could enhance the regenerative potential. The results showed that co-injecting stem cells from the apical papilla (SCAPs) and human umbilical vein endothelial cells (HUVECs) at a 1:1 ratio into the 0.04 taper fluidic microchannels, which were fabricated within 5% (w/v) GelMA hydrogel with SCAPs encapsulated, are the ideal conditions to promote angiogenesis. In the second part of this study, GelMA hydrogel scaffolds with an inbuilt microchannel loaded with SCAPs and HUVECs were transplanted into immunodeficient mice. The results showed that, four weeks after transplantation, the groups with tapers (both 0.4 and 0.6) formed pulp-like tissue that reached the entire length of the root. In contrast, the group without microchannels only generated pulp tissue to the apical and middle levels of the root canal. Co-implanting pericyte-like and endothelial cells (ECs) with extracellular matrix and angiogenic growth factors are crucial for successfully engineering a pre-vascularized tissue construct. In the third part of this study, we sought to form EC-pericyte microvessels by co-culturing HUVECs and SCAPs on perfusable 3D microfluidic chips under sequential release of growth factors. Angiopoietin 1 (Ang 1) was first encapsulated in gelatin nanoparticles (Ang 1-GNPs) and then coupled with vascular endothelial growth factor (VEGF) in GelMA hydrogel to establish a platform for temporal controlled release. The findings demonstrated that VEGF-Ang l-GNPs/GelMA hydrogel started to release VEGF before Ang 1 was released. The sprouting length and speed of vasculature were noticeably increased, while the vascular permeability was decreased, possibly due to the appropriately timed VEGF and Ang 1 release mode. In summary, we reported a novel strategy for fabricating an inbuilt tapered microchannel to generate 3D engineered tissue constructs in vitro and in vivo and a VEGF-Ang l-GNPs/GelMA hydrogel platform to regulate the release of different growth factors. The findings demonstrate the efficacy of the novel strategy and platform in accelerating angiogenesis and dental pulp regeneration. One of the viable engineering techniques to encourage angiogenesis is fabricating the built-in fluidic vascular microchannels in the middle of GelMA hydrogel. GelMA hydrogel can offer a proper milieu for developing perfusable microvascular networks of various architectural configurations, and the built-in microchannels can enhance cell survival and differentiation. Therefore, in the first part of this study, we investigated whether the varying concentrations of GelMA hydrogel scaffold in combination with the built-in fluidic vascular channels with different tapers could enhance the regenerative potential. The results showed that co-injecting stem cells from the apical papilla (SCAPs) and human umbilical vein endothelial cells (HUVECs) at a 1:1 ratio into the 0.04 taper fluidic microchannels, which were fabricated within 5% (w/v) GelMA hydrogel with SCAPs encapsulated, are the ideal conditions to promote angiogenesis. In the second part of this study, GelMA hydrogel scaffolds with an inbuilt microchannel loaded with SCAPs and HUVECs were transplanted into immunodeficient mice. The results showed that, four weeks after transplantation, the groups with tapers (both 0.4 and 0.6) formed pulp-like tissue that reached the entire length of the root. In contrast, the group without microchannels only generated pulp tissue for the apical and middle levels of the root canal. Co-implanting pericyte-like and endothelial cells (ECs) with extracellular matrix and angiogenic growth factors is crucial for successfully engineering a pre-vascularized tissue construct. In the third part of this study, we sought to form EC-pericyte microvessels by co-culturing HUVECs and SCAPs on perfusable 3D microfluidic chips under the sequential release of growth factors. Angiopoietin-1 (Ang-1) was first encapsulated in gelatin nanoparticles (Ang-1-GNPs) and then coupled with vascular endothelial growth factor (VEGF) in GelMA hydrogel to establish a platform for temporally controlled release. The findings demonstrated that VEGF-Ang l-GNPs and GelMA hydrogel started to release VEGF before Ang 1 was released. The sprouting length and speed of vasculature were noticeably increased, while the vascular permeability was decreased, possibly due to the appropriately timed VEGF and Ang 1 release modes. In summary, we reported a novel strategy for fabricating an inbuilt tapered microchannel to generate 3D engineered tissue constructs in vitro and in vivo and a VEGF-Ang l-GNPs/GelMA hydrogel platform to regulate the release of different growth factors. The findings demonstrate the efficacy of the novel strategy and platform in accelerating angiogenesis and dental pulp regeneration.