DPSC Products Accelerate Wound Healing in Diabetic Mice through Induction of SMAD Molecules.

Despite advances in diabetic wound care, many amputations are still needed each year due to their diabetic wounds, so a more effective therapy is warranted. Herein, we show that the dental pulp-derived stem cell (DPSC) products are effective in wound healing in diabetic NOD/SCID mice. Our results showed that the topical application of DPSC secretory products accelerated wound closure by inducing faster re-epithelialization, angiogenesis, and recellularization. In addition, the number of neutrophils producing myeloperoxidase, which mediates persisting inflammation, was also reduced. NFκB and its downstream effector molecules like IL-6 cause sustained pro-inflammatory activity and were reduced after the application of DPSC products in the experimental wounds. Moreover, the DPSC products also inhibited the activation of NFκB, and its translocation to the nucleus, by which it initiates the inflammation. Furthermore, the levels of TGF-ß, and IL-10, potent anti-inflammatory molecules, were also increased after the addition of DPSC products. Mechanistically, we showed that this wound-healing process was mediated by the upregulation and activation of Smad 1 and 2 molecules. In sum, we have defined the cellular and molecular mechanisms by which DPSC products accelerated diabetic wound closure, which can be used to treat diabetic wounds in the near future.

Dental pulp-derived stem cells inhibit osteoclast differentiation by secreting osteoprotegerin and deactivating AKT signalling in myeloid cells.

Osteoclasts (OCs) differentiate from the monocyte/macrophage lineage, critically regulate bone resorption and remodelling in both homeostasis and pathology. Various immune and non-immune cells help initiating activation of myeloid cells for differentiation, whereas hyper-activation leads to pathogenesis, and mechanisms are yet to be completely understood. Herein, we show the efficacy of dental pulp-derived stem cells (DPSCs) in limiting RAW 264.7 cell differentiation and underlying molecular mechanism, which has the potential for future therapeutic application in bone-related disorders. We found that DPSCs inhibit induced OC differentiation of RAW 264.7 cells when co-cultured in a contact-free system. DPSCs reduced expression of key OC markers, such as NFATc1, cathepsin K, TRAP, RANK and MMP-9 assessed by quantitative RT-PCR, Western blotting and immunofluorescence detection methods. Furthermore, quantitative RT-PCR analysis revealed that DPSCs mediated M2 polarization of RAW 264.7 cells. To define molecular mechanisms, we found that osteoprotegerin (OPG), an OC inhibitory factor, was up-regulated in RAW 264.7 cells in the presence of DPSCs. Moreover, DPSCs also constitutively secrete OPG that contributed in limiting OC differentiation. Finally, the addition of recombinant OPG inhibited OC differentiation in a dose-dependent manner by reducing the expression of OC differentiation markers, NFATc1, cathepsin K, TRAP, RANK and MMP9 in RAW 264.7 cells. RNAKL and M-CSF phosphorylate AKT and activate PI3K-AKT signalling pathway during osteoclast differentiation. We further confirmed that OPG-mediated inhibition of the downstream activation of PI3K-AKT signalling pathway was similar to the DPSC co-culture-mediated inhibition of OC differentiation. This study provides novel evidence of DPSC-mediated inhibition of osteoclastogenesis mechanisms.

Cable deployment system for unmanned ground vehicle (UGV) mobile microgrids.

An ad hoc autonomous mobile microgrid system requires electrical connections to be formed between physically separated resources. This work proposes the use of unmanned ground vehicles (UGVs) as the means to deploy the electrical cable that creates these connections. This operation requires careful control of the cabling at variable speeds to avoid entanglement with the deploying UGV or obstacles in complex outdoor environments. Searching for a product that could supply the needed control and flexibility revealed a lack of compact and low-cost options. Existing options are very heavy ( > 100  lbs) and do not supply precision in their deployment. There is no commercial off-the-shelf option available for small-scale cable deployment operations with size and weight constraints. To fulfill the application requirements and to combat this deficiency, a custom design and build of an "Adjustable Cable Management Mechanism" (ACMM) was required. This ACMM provides a low cost, compact platform for powered and controlled deployment and retraction of different-sized cable under moderate loads, utilizing Commercial Off-The-Shelf components (COTS). Employing this design has enabled a variety of tasks that require distribution of electrical or data cables to be accomplished for small-scale projects. The goal of this paper is to give detailed design specifications of the ACMM and instructions on how to recreate it and calibrate it to be useful for tethering robots in various applications such as steep terrain, internet connection through tight spaces, or electrical connection between nodes for complex microgrids.

Development of Cutaneous Wound in Diabetic Immunocompromised Mice and Use of Dental Pulp-Derived Stem Cell Product for Healing.

Chronic nonhealing wounds impact nearly 15% of Medicare beneficiaries (8.2 million) in the United States costing $28-$32 billion annually. Despite advancement in wound management, approximately 8% of diabetic Medicare beneficiaries have a foot ulcer and 1.8% will have an amputation. The development of a regenerative approach is warranted to save these before-mentioned amputations. To this extent, herein, we describe the detailed methods in generating a type 1 diabetes mellitus (T1DM) condition in immunocompromised mice, inducing cutaneous wound, and application of dental pulp stem cell-derived secretory products for therapeutic assessment. This model helps in evaluating the efficacy of stem cell-based therapy and helps with the investigation of involved mechanisms in impaired cutaneous wound healing caused by hyperglycemic stress due to type 1 diabetes.

KLF2 regulates dental pulp-derived stem cell differentiation through the induction of mitophagy and altering mitochondrial metabolism.

To define the regulatory role of Kruppel-like factor 2 (KLF2) during osteoblast (OB) differentiation of dental pulp-derived stem cell (DPSC)s, herein, we show that the levels of KLF2 and autophagy-related molecules were significantly increased in differentiated cells. Gain-of-function and loss-of-function approaches of KLF2 confirmed that KLF2 modulated autophagic and OB differentiation-related molecules. In addition, knockdown of the autophagic molecule (ATG7 or BECN1) in DPSCs resulted in reduced levels of KLF2 and OB differentiation-related molecules. Conversely, the induction of autophagy increased levels of KLF2 and OB differentiation-related molecules. Moreover, OB differentiation induced mitophagy and mitochondrial membrane potential-related molecules. In addition, OB differentiation reduced the generation of total and mitochondrial ROS productions and induced intracellular Ca2+ production. Measurements of glycolysis and oxidative phosphorylation simultaneously in live cells revealed that OB differentiation decreased the oxygen consumption rate, which is an indicator of mitochondrial respiration and reduced the level of ATP production. Furthermore, flux analysis also revealed that OB differentiation increased the extracellular acidification rate (ECAR) in the non-glycolytic acidification, and the glycolytic capacity conditions, increasing the lactate production and reducing the metabolic activity of the cells. Thus, a metabolic shift from mitochondrial respiration to the glycolytic pathway was observed during OB differentiation. Finally, chromatin immunoprecipitation (ChIP) analysis confirmed that the KLF2 and active epigenetic marks (H3K27Ac and H3K4me3) were upregulated in the promoter region of ATG7 during OB differentiation. These results provide evidence that the mitophagy process is important during OB differentiation, and KLF2 critically regulates it.

Ferutinin directs dental pulp-derived stem cells towards the osteogenic lineage by epigenetically regulating canonical Wnt signaling.

Osteoporosis is a silent systemic disease that causes bone deterioration, and affects over 10 million people in the US alone. This study was undertaken to develop a potential stem cell therapy for osteoporosis. We have isolated and expanded human dental pulp-derived stem cells (DPSCs), characterized them, and confirmed their multipotential differentiation abilities. Stem cells often remain quiescent and require activation to differentiate and function. Herein, we show that ferutinin activates DPSCs by modulating the Wnt/ß-catenin signaling pathway and key osteoblast-secreted proteins osteocalcin and collagen 1A1 both mRNA and protein levels. To confirm that ferutinin modulates the Wnt pathway, we inhibited glycogen synthase kinase 3 (GSK3) and found that protein expression patterns were similar to those found in ferutinin-treated DPSCs. To evaluate the role of ferutinin in epigenetic regulation of canonical Wnt signaling, the pathway molecules Wnt3a and Dvl3 were analyzed using chromatin immunoprecipitation (ChIP)-quantitative PCR approaches. We confirmed that active marks of both H3K9 acetylation and H3K4 trimethylation were significantly enhanced in the promoter sites of the WNT3A and DVL3 genes in DPSCs after addition of ferutinin. These data provide evidence that ferutinin activates and promotes osteogenic differentiation of DPSCs, and could be used as an inducer as a potentially effective stem cell therapy for osteoporosis.