Silencing VEGFR-2 Hampers Odontoblastic Differentiation of Dental Pulp Stem Cells.

Dental pulp stem cells (DPSCs) are a source of postnatal stem cells essential for maintenance and regeneration of dentin and pulp tissues. Previous in vivo transplantation studies have shown that DPSCs are able to give rise to odontoblast-like cells, form dentin/pulp-like structures, and induce blood vessel formation. Importantly, dentin formation is closely associated to blood vessels. We have previously demonstrated that DPSC-induced angiogenesis is VEGFR-2-dependent. VEGFR-2 may play an important role in odontoblast differentiation of DPSCs, tooth formation and regeneration. Nevertheless, the role of VEGFR-2 signaling in odontoblast differentiation of DPSCs is still not well understood. Thus, in this study we aimed to determine the role of VEGFR-2 in odontoblast differentiation of DPSCs by knocking down the expression of VEGFR-2 in DPSCs and studying their odontoblast differentiation capacity in vitro and in vivo. Isolation and characterization of murine DPSCs was performed as previously described. DPSCs were induced by VEGFR-2 shRNA viral vectors transfection (MOI = 10:1) to silence the expression of VEGFR-2. The GFP+ expression in CopGFP DPSCs was used as a surrogate to measure the efficiency of transfection and verification that the viral vector does not affect the expression of VEGFR-2. The efficiency of viral transfection was shown by significant reduction in the levels of VEGFR-2 based on the Q-RT-PCR and immunofluorescence in VEGFR-2 knockdown DPSCs, compared to normal DPSCs. VEGFR-2 shRNA DPSCs expressed not only very low level of VEGFR-2, but also that of its ligand, VEGF-A, compared to CopGFP DPSCs in both transcriptional and translational levels. In vitro differentiation of DPSCs in osteo-odontogenic media supplemented with BMP-2 (100 ng/ml) for 21 days demonstrated that CopGFP DPSCs, but not VEGFR-2 shRNA DPSCs, were positive for alkaline phosphatase (ALP) staining and formed mineralized nodules demonstrated by positive Alizarin Red S staining. The expression levels of dentin matrix proteins, dentin matrix protein-1 (Dmp1), dentin sialoprotein (Dspp), and bone sialoprotein (Bsp), were also up-regulated in differentiated CopGFP DPSCs, compared to those in VEGFR-2 shRNA DPSCs, suggesting an impairment of odontoblast differentiation in VEGFR-2 shRNA DPSCs. In vivo subcutaneous transplantation of DPSCs with hydroxyapatite (HAp/TCP) for 5 weeks demonstrated that CopGFP DPSCs were able to differentiate into elongated and polarized odontoblast-like cells forming loose connective tissue resembling pulp-like structures with abundant blood vessels, as demonstrated by H&E, Alizarin Red S, and dentin matrix staining. On the other hand, in VEGFR-2 shRNA DPSC transplants, odontoblast-like cells were not observed. Collagen fibers were seen in replacement of dentin/pulp-like structures. These results indicate that VEGFR-2 may play an important role in dentin regeneration and highlight the potential of VEGFR-2 modulation to enhance dentin regeneration and tissue engineering as a promising clinical application.

Tunable electroconductive decellularized extracellular matrix hydrogels for engineering human cardiac microphysiological systems.

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) offer tremendous potential when used to engineer human tissues for drug screening and disease modeling; however, phenotypic immaturity reduces assay reliability when translating in vitro results to clinical studies. To address this, we have developed hybrid hydrogels comprised of decellularized porcine myocardial extracellular matrix (dECM) and reduced graphene oxide (rGO) to provide a more instructive microenvironment for proper cell and tissue development. A tissue-specific protein profile was preserved post-decellularization, and through the modulation of rGO content and degree of reduction, the mechanical and electrical properties of the hydrogels could be tuned. Engineered heart tissues (EHTs) generated using dECM-rGO hydrogel scaffolds and hiPSC-derived cardiomyocytes exhibited significantly increased twitch forces and had increased expression of genes that regulate contractile function. Improvements in various aspects of electrophysiological function, such as calcium-handling, action potential duration, and conduction velocity, were also induced by the hybrid biomaterial. dECM-rGO hydrogels could also be used as a bioink to print cardiac tissues in a high-throughput manner, and these tissues were utilized to assess the proarrhythmic potential of cisapride. Action potential prolongation and beat interval irregularities was observed in dECM-rGO tissues at clinical doses of cisapride, indicating that the enhanced electrophysiological function of these tissues corresponded well with a capability to produce physiologically relevant drug responses.

Conductive Silk-Polypyrrole Composite Scaffolds with Bioinspired Nanotopographic Cues for Cardiac Tissue Engineering.

We report on the development of bioinspired cardiac scaffolds made from electroconductive acid-modified silk fibroin-poly(pyrrole) (AMSF+PPy) substrates patterned with nanoscale ridges and grooves reminiscent of native myocardial extracellular matrix (ECM) topography to enhance the structural and functional properties of cultured human pluripotent stem cells (hPSC)-derived cardiomyocytes. Nanopattern fidelity was maintained throughout the fabrication and functionalization processes, and no loss in conductive behavior occurred due to the presence of the nanotopographical features. AMSF+PPy substrates were biocompatible and stable, maintaining high cell viability over a 21-day culture period while displaying no signs of PPy delamination. The presence of anisotropic topographical cues led to increased cellular organization and sarcomere development, and electroconductive cues promoted a significant improvement in the expression and polarization of connexin 43 (Cx43), a critical regulator of cell-cell electrical coupling. The combination of biomimetic topography and electroconductivity also increased the expression of genes that encode key proteins involved in regulating the contractile and electrophysiological function of mature human cardiac tissue.

Harnessing Sphingosine-1-Phosphate Signaling and Nanotopographical Cues To Regulate Skeletal Muscle Maturation and Vascularization.

Despite possessing substantial regenerative capacity, skeletal muscle can suffer from loss of function due to catastrophic traumatic injury or degenerative disease. In such cases, engineered tissue grafts hold the potential to restore function and improve patient quality of life. Requirements for successful integration of engineered tissue grafts with the host musculature include cell alignment that mimics host tissue architecture and directional functionality, as well as vascularization to ensure tissue survival. Here, we have developed biomimetic nanopatterned poly(lactic-co-glycolic acid) substrates conjugated with sphingosine-1-phosphate (S1P), a potent angiogenic and myogenic factor, to enhance myoblast and endothelial maturation. Primary muscle cells cultured on these functionalized S1P nanopatterned substrates developed a highly aligned and elongated morphology and exhibited higher expression levels of myosin heavy chain, in addition to genes characteristic of mature skeletal muscle. We also found that S1P enhanced angiogenic potential in these cultures, as evidenced by elevated expression of endothelial-related genes. Computational analyses of live-cell videos showed a significantly improved functionality of tissues cultured on S1P-functionalized nanopatterns as indicated by greater myotube contraction displacements and velocities. In summary, our study demonstrates that biomimetic nanotopography and S1P can be combined to synergistically regulate the maturation and vascularization of engineered skeletal muscles.