Dental pulp infection and necrosis are widespread diseases. Conventional endodontic treatments result in a devitalized and weakened tooth. In this work, we synthesized novel star-shaped polymer to self-assemble into unique nanofibrous spongy microspheres (NF-SMS), which were used to carry human dental pulp stem cells (hDPSCs) into the pulp cavity to regenerate living dental pulp tissues. It was found that NF-SMS significantly enhanced hDPSCs attachment, proliferation, odontogenic differentiation and angiogenesis, as compared to control cell carriers. Additionally, NF-SMS promoted vascular endothelial growth factor (VEGF) expression of hDPSCs in a 3D hypoxic culture. Hypoxia-primed hDPSCs/NF-SMS complexes were injected into the cleaned pulp cavities of rabbit molars for subcutaneous implantation in mice. After 4 weeks, the hypoxia group significantly enhanced angiogenesis inside the pulp chamber and promoted the formation of ondontoblast-like cells lining along the dentin-pulp interface, as compared to the control groups (hDPSCs alone group, NF-SMS alone group, and hDPSCs/NF-SMS group pre-cultured under normoxic conditions). Furthermore, in an in situ dental pulp repair model in rats, hypoxia-primed hDPSCs/NF-SMS were injected to fully fill the pulp cavity and regenerate pulp-like tissues with a rich vasculature and a histological structure similar to the native pulp.

We describe in this paper the structure-based design of a general class of heterocyclic mechanism-based inhibitors of the serine proteinases that embody in their structure a novel peptidomimetic scaffold (1,2,5-thiadiazolidin-3-one 1,1-dioxide). Sulfone derivatives of this class (I) were found to be time-dependent, potent, and highly efficient irreversible inhibitors of human leukocyte elastase, cathepsin G, and proteinase 3. The partition ratios for a select number of inhibitors were found to range between 0 and 1. We furthermore demonstrate that these inhibitors exhibit remarkable enzyme selectivity that is dictated by the nature of the P1 residue and is consistent with the known substrate specificity reported for these enzymes. Thus, inhibitors with small hydrophobic side chains were found to be effective inhibitors of elastase, those with aromatic side chains of cathepsin G, and those with a basic side chain of bovine trypsin. Taken together, the findings cited herein reveal the emergence of a general class of stable mechanism-based inhibitors of the serine proteinases which can be readily synthesized using amino acid precursors. Biochemical and high-field NMR studies show that the interaction of this class of inhibitors with a serine proteinase results in the formation of a stable acyl complex(es) and the release of benzenesulfinate, formaldehyde, and a low molecular weight heterocycle. The data are consistent with initial formation of a Michaelis-Menten complex, acylation of Ser195, and tandem loss of the leaving group. The initial HLE-inhibitor complex reacts with water generating formaldehyde and a stable HLE-inhibitor complex. Whether the initial HLE-inhibitor complex also reacts with His57 to form a third complex is not known at this point. The desirable salient parameters associated with this class of inhibitors, including the expeditious generation of structurally diverse libraries of inhibitors based on I, suggest that this class of mechanism-based inhibitors is of general applicability and can be used in the development of inhibitors of human and viral serine proteinases of clinical relevance.

Pain caused by acute pulpitis (AP) is a common symptom in clinical settings. However, its underlying mechanisms have largely remained unknown. Using AP model, we demonstrated that dental injury caused severe pulp inflammation with up-regulated serum IL-1β. Assessment from head-withdrawal reflex thresholds (HWTs) and open-field test demonstrated nociceptive response at 1 day post injury. A consistent up-regulation of Toll-like receptor 4 (TLR4) in the trigeminal ganglion (TG) ipsilateral to the injured pulp was found; and downstream signaling components of TLR4, including MyD88, TRIF and NF-κB, and cytokines such as TNF-α and IL-1β, were also increased. Retrograde labeling indicated that most TLR4 positve neuron in the TG innnervated the pulp and TLR4 immunoreactivity was mainly in the medium and small neurons. Double labeling showed that the TLR4 expressing neurons in the ipsilateral TG were TRPV1 and CGRP positive, but IB4 negative. Furthermore, blocking TLR4 by eritoran (TLR4 antagonist) in TGs of the AP model significantly down-regulated MyD88, TRIF, NF-κB, TNF-α and IL-1β production and behavior of nociceptive response. Our findings suggest that TLR4 signaling in TG cells, particularly the peptidergic TRPV1 neurons, plays a key role in AP-induced nociception, and indicate that TLR4 signaling could be a potential therapeutic target for orofacial pain.

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