Ginseng-derived exosome-like nanovesicles extracted by sucrose gradient ultracentrifugation to inhibit osteoclast differentiation.

Plant-derived extracellular nanovesicles contain RNA and proteins with unique and diverse pharmacological mechanisms. The extracellular nanovesicles encapsulating plant extracts resemble exosomes as they have a round, lipid bilayer morphology. Ginseng is anti-inflammatory, anti-cancer, immunostimulant, and osteogenic/anti-osteoporotic. Here, we confirmed that ginseng-derived extracellular nanovesicles (GDNs) inhibit osteoclast differentiation and elucidated the associated molecular mechanisms. We isolated GDNs by centrifugation with a sucrose gradient. We measured their dynamic light scattering and zeta potentials and examined their morphology by transmission electron microscopy. We used bone marrow-derived macrophages (BMMs) to determine the potential cytotoxicity of GDNs and establish their ability to inhibit osteoclast differentiation. The GDNs treatment maintained high BMM viability and proliferation whilst impeding osteoclastogenesis. Tartrate-resistant acid phosphatase and F-actin staining revealed that GDNs at concentrations >1 µg mL-1 strongly hindered osteoclast differentiation. Moreover, they substantially suppressed the RANKL-induced IκBα, c-JUN n-terminal kinase, and extracellular signal-regulated kinase signaling pathways and the genes regulating osteoclast maturation. The GDNs contained elevated proportions of Rb1 and Rg1 ginsenosides and were more effective than either of them alone or in combination at inhibiting osteoclast differentiation. In vivo bone analysis via microcomputerized tomography, bone volume/total volume ratios, and bone mineral density and bone cavity measurements demonstrated the inhibitory effect of GDNs against osteoclast differentiation in lipopolysaccharide-induced bone resorption mouse models. The results of this work suggest that GDNs are anti-osteoporotic by inhibiting osteoclast differentiation and are, therefore, promising for use in the clinical prevention and treatment of bone loss diseases.

Effects of Scoparone on differentiation, adhesion, migration, autophagy and mineralization through the osteogenic signalling pathways.

Scoparone (SCOP), an active and efficient coumarin compound derived from Artemisia capillaris Thunb, has been used as a traditional Chinese herbal medicine. Herein, we investigated the effects of SCOP on the osteogenic processes using MC3T3-E1 pre-osteoblasts in in vitro cell systems. SCOP (C11 H10 O4 , > 99.17%) was purified and identified from A. capillaries. SCOP (0.1 to 100 µM concentrations) did not have cytotoxic effects in pre-osteoblasts; however, it promoted alkaline phosphatase (ALP) staining and activity, and mineralized nodule formation under early and late osteogenic induction. SCOP elevated osteogenic signals through the bone morphogenetic protein 2 (BMP2)-Smad1/5/8 pathway, leading to the increased expression of runt-related transcription factor 2 (RUNX2) with its target protein, matrix metallopeptidase 13 (MMP13). SCOP also induced the non-canonical BMP2-MAPKs pathway, but not the Wnt3a-ß-catenin pathway. Moreover, SCOP promoted autophagy, migration and adhesion under the osteogenic induction. Overall, the findings of this study demonstrated that SCOP has osteogenic effects associated with cell differentiation, adhesion, migration, autophagy and mineralization.

The Incidence of Confounding Factors in Patients With Diabetes Mellitus Hospitalized for Diabetic Foot Ulcers.

INTRODUCTION: Uncontrolled deformity, deep infection, and/or ischemia-hypoxia are highly associated with healing challenges of diabetic foot ulcers (DFUs). This paper reports the occurrences of these factors that the authors label the "Troublesome Triad" (TT) in a prospective series of 62 patients with diabetes mellitus (DM), who were hospitalized because of their DFUs. MATERIALS AND METHODS: With Institutional Review Board approval, the authors gathered data in a prospective series of patients hospitalized because of lower extremity wounds. From this data, they analyzed the DFU cohort for the incidence of each of the components of the TT. The severity of the wound was graded with the authors' 0 to 10 Wound Score in the patients who had components of the TT and compared with those who did not. RESULTS: One or more components of the TT were observed in 57 patients (91.9%). As the number of confounders increased, mean Wound Scores decreased from 5.2 for 1 confounder to 2.9 for 3 confounders (P = 0.003). Most patients had 1 or 2 confounders (38.7% and 45.2%, respectively), while only 5 (8.1%) patients had all 3 confounders. Unresolved infection was the major confounder in 38 (61.3%) patients, uncontrolled deformity in 31 (50.0%), and ischemia-hypoxia in 26 (41.9%). CONCLUSION: For those patients with DM who were hospitalized because of DFUs, confounders that require remedial interventions were present in more than 90% of patients. Recognition and management of the TT eliminates wasteful uses of resources in an attempt to heal lower extremity wounds in patients with DM where the confounders need to be addressed first.

The validity of transcutaneous oxygen measurements in predicting healing of diabetic foot ulcers.

INTRODUCTION: In 2002 Fife and Strauss (Fife, et al. Wound Rep Reg, 10:198-207; Strauss, et al. Foot Ankle Intl, 23:933-937) studied the predictability of transcutaneous oxygen measurements (TCOMs) for healing diabetic foot ulcers (DFUs). This paper analyzes the validity of the two studies and combines their information to predict which DFU will heal with adjunctive hyperbaric oxygen (HBO2) treatments. METHODS: A statistical review of the Fife and Strauss papers was performed. The numbers presented in the papers were subjected to analyses to compare like by like data as well as test for p-values and odds ratios for predicting healing of DFUs with HBO2. RESULTS: In the Strauss paper 143 subjects were studied in retrospective and prospective series. In those TCOMs which exceed 200 mmHg with HBO2 healing occurred in 87.5% even if the room air TCOM was ⟨ 30 mmHg (p ⟨ 0.001). The Fife paper studied retrospectively a subset of 221 patients who had TCOMs with HBO2. Failure rates for healing decreased progressively from 35.7% to 14.3%, with TCOMs grouped in 100-mmHg increments from 200 mmHg to 699 mmHg. This resulted in absence of statistical significance for any 100-mmHg range over 200 mmHg with HBO2 due to the small number of subjects for each 100-mmHg grouping. CONCLUSIONS: Although differences exist between the study designs, each complements the other. If TCOMs exceed 200 mmHg with HBO2, both authors observed that almost 90% of DFUs healed regardless of the room air readings when HBO2 was used as an adjunct to management.

Safflower seed extract inhibits osteoclast differentiation by suppression of the p38 mitogen-activated protein kinase and IκB kinase activity.

Safflower seed has been reported to have a protective effect against bone loss diseases. However, the precise molecular mechanisms underlying the inhibitory effect of safflower seed in osteoclast differentiation remain unclear. In this study, we investigated the inhibitory action of safflower seed extract (SSE) on the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in cultured mouse-derived bone marrow macrophages (BMMs). We found that SSE significantly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in BMMs without cytotoxicity. The gene expressions of nuclear factor of activated T-cells (NFATc1) and TRAP, which are genetic markers of osteoclast differentiation, were substantially decreased by SSE in a dose-dependent manner. Also, SSE diminished RANKL-mediated intracellular reactive oxygen species (ROS) generation on osteoclastogenesis in a dose-dependent manner. The SSE thereafter suppressed RANKL-induced p38 mitogen-activated protein kinase and IκBα kinase signalling activities which were activated by ROS generation for osteoclastogenesis. Additionally, SSE was found to decrease RANKL-induced actin ring formation, which is required for bone resorption activity. Taken together, our results suggest that SSE acts as a RANKL-induced osteoclastogenesis inhibitor by suppression of ROS generation. This induces a remarkable suppression of the p38 and IκBα kinase pathways, thereby suppressing the gene expression of NFATc1 in osteoclast precursors.