Alginate/laponite hydrogel microspheres co-encapsulating dental pulp stem cells and VEGF for endodontic regeneration

Pulp necrosis arrests root development in injured immature permanent teeth, which may result in tooth loss. However, dental pulp regeneration and promotion of root development remains challenging. We show that implantation of autologous tooth stem cells from deciduous teeth regenerated dental pulp with an odontoblast layer, blood vessels, and nerves in two animal models. These results prompted us to enroll 40 patients with pulp necrosis after traumatic dental injuries in a randomized, controlled clinical trial. We randomly allocated 30 patients to the human deciduous pulp stem cell (hDPSC) implantation group and 10 patients to the group receiving traditional apexification treatment. Four patients were excluded from the implantation group due to loss at follow-up (three patients) and retrauma of the treated tooth (one patient). We examined 26 patients (26 teeth) after hDPSC implantation and 10 patients (10 teeth) after apexification treatment. hDPSC implantation, but not apexification treatment, led to regeneration of three-dimensional pulp tissue equipped with blood vessels and sensory nerves at 12 months after treatment. hDPSC implantation increased the length of the root (P < 0.0001) and reduced the width of the apical foramen (P < 0.0001) compared to the apexification group. In addition, hDPSC implantation led to regeneration of dental pulp tissue containing sensory nerves. To evaluate the safety of hDPSC implantation, we followed 20 patients implanted with hDPSCs for 24 months and did not observe any adverse events. Our study suggests that hDPSCs are able to regenerate whole dental pulp and may be useful for treating tooth injuries due to trauma.

We describe a protocol for easy isolation and culture of human umbilical vein endothelial cells (HUVECs) to supply every researcher with a method that can be applied in cell biology laboratories with minimum equipment. Endothelial cells (ECs) are isolated from umbilical vein vascular wall by a collagenase treatment, then seeded on fibronectin-coated plates and cultured in a medium with Earles' salts and fetal calf serum (FCS), but without growth factor supplementation, for 7 days in a 37 degrees C-5% CO2 incubator. Cell confluency can be monitored by phase-contrast microscopy; ECs can be characterized using cell surface or intracellular markers and checked for contamination. Various protocols can be applied to HUVECs, from simple harvesting to a particular solubilization of proteins for proteomic analysis.

Contractile myocytes provide a test of the hypothesis that cells sense their mechanical as well as molecular microenvironment, altering expression, organization, and/or morphology accordingly. Here, myoblasts were cultured on collagen strips attached to glass or polymer gels of varied elasticity. Subsequent fusion into myotubes occurs independent of substrate flexibility. However, myosin/actin striations emerge later only on gels with stiffness typical of normal muscle (passive Young's modulus, E ∼12 kPa). On glass and much softer or stiffer gels, including gels emulating stiff dystrophic muscle, cells do not striate. In addition, myotubes grown on top of a compliant bottom layer of glass-attached myotubes (but not softer fibroblasts) will striate, whereas the bottom cells will only assemble stress fibers and vinculin-rich adhesions. Unlike sarcomere formation, adhesion strength increases monotonically versus substrate stiffness with strongest adhesion on glass. These findings have major implications for in vivo introduction of stem cells into diseased or damaged striated muscle of altered mechanical composition.