Bioengineering vascularized and innervated dental pulp construct

Abstract

Regenerative endodontic therapy offers an alternative strategy for the management of dental pulp and periapical diseases. However, this novel method is hampered by the incapacity to attain a sufficient blood supply following implantation due to the anatomical confinement of the root canal system. Achieving adequate angiogenesis within bioengineered tissue and instant anastomosis with host circulation after implantation is crucial for ensuring successful regenerative treatment. To facilitate vascularization and accelerate anastomosis, various approaches, such as the recruitment of dental stem cells (i.e. DPSCs, SHEDs and SCAPs), delivery of pro-angiogenic factors (i.e. VEGF, bFGF, PDGF-BB, ANG1, TGF-β, IGF-1), and the combination of stem cells, scaffolds and signaling molecules, have been explored. To bioengineer vascularized and innervated dental pulp, we synthesized and characterized photocrosslinkable GelMA-based hydrogels, which can be easily crosslinked by dental curing light. The physical and mechanical properties of GelMA hydrogels were precisely manipulated and optimized for maintaining NSCs viability and proliferation. It was demonstrated that a lower concentration of GelMA hydrogel exhibits a higher swelling ration and larger pore size but a lower elastic modulus. Soft GelMA hydrogel substrate favorably supports NSCs proliferation. Furthermore, survival population and neurite outgrowth of trigeminal neuron embedded in soft hydrogels are superior to these features in stiff hydrogels, and neurons exhibit longer neurites in 3D culture. PDMS scaffold with pre-fabricated micro-channel by GelMA hydrogel enhances trigeminal neurite migration. Semaphorins are expressed in various stages during development of the tooth germ. Sema4D, a member of the semaphorin family, exerts dramatic regulation of angiogenesis in a manner analogous to VEGF. In this study, the effects of Sema4D on osteogenic differentiation were investigated. The results showed that 1) Sema4D significantly inhibited ALP activity and mineralization of DPSCs and 2) Sema4D suppressed osteogenic-related genes and protein expression of DPSCs. Additionally, Sema4D upregulated the VEGF gene and protein level in DPSCs. Vessel-like tube formation assays further indicated that the supernatants of Sema4D-treated DPSCs enhanced endothelial cell vessel-like network formation. To further investigate whether Sema4D might induce DPSCs endotheliogenesis, angiogenic and endothelial specific genes were analyzed. We found that Sema4D treatment of DPSCs triggered phosphorylation of AKT and ERK1/2, which was accompanied by an upregulation of endothelial differentiation-related genes (VEGFR1, VEGFR2, CD31 and vWF) and angiogenesis-related genes (HIF-1⍺, ANG1 and ANGPTL4). VEGF, HIF-1⍺, and ANGPTL4 protein expression was also increased. In conclusion, GelMA hydrogel properties can be tailored by controlling the concentration of GelMA. Soft GelMA hydrogel maintains NSCs viability and proliferation. PDMS scaffolds with pre-fabricated microchannels by GelMA hydrogel enhances trigeminal neurite migration. Sema4D/plexin-B1 signaling enhances the angiogenic potential but inhibits the osteogenic differentiation of DPSCs. The axis of Sema4D, ANGPTL4 and HIF-1⍺ plays a critical role in the regulating the endothelial differentiation of DPSCs via the phosphorylation of AKT and ERK1/2.